Prion free nanoparticle compositions and methods of making thereof

ABSTRACT

The present invention provides prion-free compositions comprising nanoparticles comprising albumin and substantially water insoluble drugs. Also provided are methods of making prion-free compositions and methods of removing prion proteins from the nanoparticle compositions. Methods of using the compositions, as well as kits useful for carrying out the methods are also provided.

RELATED APPLICATIONS

The application claims priority benefit to U.S. Provisional PatentApplication No. 61/169,665, filed Apr. 15, 2009, and U.S. ProvisionalPatent Application No. 61/238,052, filed Aug. 28, 2009, the content ofeach of which is incorporated herein in their entirety.

TECHNICAL FIELD

The present invention relates to prion-free compositions comprisingnanoparticles comprising albumin and substantially water insolubledrugs, methods of making, and methods of using thereof.

BACKGROUND

Prion diseases, also known as transmissible spongiform encephalopathies(TSEs), are a group of fatal, transmittable neurodegenerative diseases.Specific examples of TSE include scrapie, which affects sheep and goats,bovine spongiform encephalopathy (B SE), transmissible minkencephalopathy, feline spongiform encephalopathy and chronic wastingdisease (CWD). In humans, TSE diseases may present themselves as kuru,Creutzfeldt-Jekob disease (CJD), Gerstmann-Straussler-Scheinker Syndrome(GSS), fatal insomnia and variant Creutzfeldt-Jekob disease (vCJD). vCJDemerged in humans as a result of the BSE epidemic in Britain and is mostprobably caused by the consumption of food products derived from cattleinfected with BSE or “mad cow disease.” (Will et al. (1996) Lancet347:921-925) Because the incubation period for the orally contracteddisease may be more than 20 years in human, the true incidence of vCJDmay not become apparent for many years.

In addition to ingestion of infected products of bovine origin, bloodtransfusion and organ transplantation represent another mode oftransmission of vCJD among humans (Brown et al. (1998) Transfusion38:810-816; Diringer et al. (1984) Archives of Virology 82:105-109;Manuelidis et al. (1978) Nature 271:778-779). Major concerns were raisedsince the mid-1990s that vCJD can be transmitted through bloodtransfusion or other blood products from TSE-infected individuals. Theseindividuals may be asymptomatic during the long pre-clinical andincubation phase of vCJD, and blood obtained from such donors may beable to transmit the disease to persons receiving the blood or bloodproducts derived from the donor.

There are so far at least four reported human cases of blood transfusionacquired vCJD in the United Kingdom. Of 64 people who received wholeblood from 22 donors, 4 people went on to develop vCJD. In the firstincidence, the recipient became ill 7 years after receiving red cellsfrom the donor who remained asymptomatic and only showed signs of vCJDuntil 3 years after the donation (Llewely et al. (2004) Lancet363:417-421). In the second incidence, the donor died of vCJD two yearsafter donation, and the recipient died of an aneurysm (not vCJD) 5 yearsafter donation (Peden et al. (2004) Lancet 364:527-529). On autopsy ofthe recipient, PrPsc was present in lymph node and spleen, but not thebrain. In the third incidence, the recipient died of vCJD seven and halfyears after transfusion from a donor who developed vCJD 20 months afterthe donation (Wroe et al. (2006) Lancet 368:2061-2067). The fourthincident occurred in a recipient eight and half years after atransfusion from the same donor in the third case (Health ProtectionAgency-Health Protection Report, (2007) Vol 1, No 3, 26. Available at:http://www.hpa.org.uk/hpdarchives/2007/news2007/news0307.htm).

A common feature of all prion diseases is the conversion of the normalcellular prion protein (PrPc) into an abnormal isoform (PrPsc). Thedifference between PrPc and PrPsc are believed to be purelyconformational, with PrPc having primarily alpha-helical structures andPrPsc having primarily beta sheets that frequently assemble to formaggregates. PrPsc acts as a template to induce normal protein moleculesto convert into the same abnormal isoform, which then in turn covertmore PrPc into PrPsc (Prusiner et al. (1998) Proc. Natl. Acad. Sci. USA95:13363-13383). This autocatalytic process leads to exponentialformation of neurotoxic PrPsc aggregates (Aguzzi et al. (2007) Nat RevMol. Cell Biol. 8:552-561). Prion protein ligands and uses thereof havebeen described in WO04/050851, WO06/010915, WO04/090102, andWO06/044459.

Studies have shown that the earliest appearance of prion infectivity inthe blood may occur during the early stage of the incubation period ofthe disease (Brown et al. (2006) Blood infectivity in the transmissiblespongiform encephalopathies. Chapter 4 In: Turner M L, ed. 95-118).Because it can be a long time before the onset of disease symptoms,silently infected individuals may still be considered as healthy activeblood donors. Furthermore, some individuals may be permanently ortransiently infected without developing the disease. It is thusdifficult if not impossible to ensure that sources of blood for bloodderived products are prion free.

Albumin-based nanoparticle compositions have been developed as a drugdelivery system for delivering substantially water insoluble drugs. See,for example, U.S. Pat. Nos. 5,916,596; 6,506,405; 6,749,868, and6,537,579 and also in U.S. Pat. Pub. Nos. 2005/0004002 and 2007/0082838.The albumin-based nanoparticle technology utilizes the unique naturalproperties of the protein albumin to transport and deliver substantiallywater insoluble drugs to the site of disease. These nanoparticles arereadily incorporated into the body's own transport processes and areable to exploit the tumors' attraction to albumin, enabling the deliveryof higher concentrations of the active drug to the target site. Inaddition, the albumin-based nanoparticle technology offers the abilityto improve a drug's solubility by avoiding the need for toxic chemicals,such as solvents, in the administration process, thus potentiallyimproving safety through the elimination of solvent-related sideeffects.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention in one aspect provides prion-free nanoparticlecompositions (such as pharmaceutical compositions). In some embodiments,there is provided a composition comprising nanoparticles comprisingalbumin and a substantially water insoluble pharmacologically activeagent, wherein the composition is substantially free of a prion protein.In some embodiments, the composition is sterile. In some embodiments,the composition is sterile filterable. In some embodiments, thecomposition further comprises a pharmaceutically acceptable carrier.

In some embodiments, the composition has less than about 100 fg/ml prionprotein. In some embodiments, the composition has a prion infectivity ofless than about 10 IU-ic/ml. In some embodiments, the composition has aprion infectivity of less than about 1 LD₅₀/ml.

In some embodiments, the composition does not show the presence of aprion protein based on a protein misfolding cyclic amplification (PMCA)assay. In some embodiments, the composition does not show the presenceof a prion protein based on an IPCR assay. In some embodiments, thecomposition has a prion infectivity of less than about 10 IU-ic/ml anddoes not show the presence of a prion protein based on a PMCA assay. Insome embodiments, the composition has a prion infectivity of less thanabout 10 IU-ic/ml and does not show the presence of a prion proteinbased on an IPRC assay.

The compositions described herein are generally substantially free ofPrPsc. In some embodiments, the composition is also substantially freeof PrPc. In some embodiments, the molar ratio of PrPsc and PrPc in thecomposition is no greater than about 1:1, such as no great than aboutany one of 1:10, 1:100, 1:1000, 1:10000, or 1:100000.

The composition described herein in some embodiments contains an amount(for example, a trace amount) of substances introduced during aprion-removal process. For example, in some embodiments, there isprovided a composition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe composition is substantially free of a prion protein, and whereinthe composition comprises an amount (for example, a trace amount) of aligand capable of binding to a prion protein. In some embodiments, thereis provided a composition comprising nanoparticles comprising albuminand a substantially water insoluble pharmacologically active agent,wherein the composition is substantially free of a prion protein, andwherein the composition comprises an amount (for example, a traceamount) of a supporting material (such as supporting material describedherein, including a resin). In some embodiments, there is provided acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe composition is substantially free of a prion protein, and whereinthe composition comprises an amount of a PRDT resin (for example, atrace amount of a PRDT resin). In some embodiments, there is provided acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe composition is substantially free of a prion protein, and whereinthe composition comprises an amount of a DVR resin (for example, a traceamount of a DVR resin).

In some embodiments, the level of an albumin stabilizer in thecomposition is less than that of a composition wherein the albumin hasnot been cleared by a prion-removal process. These albumin stabilizersinclude, for example, N-acetyl tryptophanate and sodium caprylate.

In some embodiments, the composition is bioequivalent to a compositionwherein the albumin has not been cleared by a prion-removal process.

In some embodiments, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, wherein the albumin in the compositionwas obtained by a method comprising a prion-removal process, said prionremoval process comprising contacting an initial albumin compositionwith a ligand capable of binding to a prion protein. In someembodiments, the prion-removal process further comprises removing saidligand and proteins bound thereto from said albumin composition.

In some embodiments, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, wherein the albumin in the compositionwas obtained by a method comprising: a) contacting an initial albumincomposition with a ligand capable of binding to a prion protein to causeformation of a complex between the ligand and a prion protein, and b)removing the complex from the initial composition.

In some embodiments, the initial albumin composition is a blood derivedproduct. In some embodiments, the initial albumin composition is analbumin composition prepared from a body fluid (such as blood). In someembodiments, the albumin is human serum albumin

In some embodiments, the ligand is a peptide (such as any peptidesprovided in Table 1). In some embodiments, the ligand is an antibodyrecognizing a prion protein. In some embodiments, the ligand is achemical compound (such as triazine-based compounds). In someembodiments, the ligand comprises an amino group, such as an amino groupon an amino resin.

The ligand can be attached to a supporting material, including, forexample, column, bead, matrix, filter, and membrane.

In another aspect, there are provided methods of producing prion-freenanoparticle compositions. For example, in some embodiments, there isprovided a method of producing a composition comprising nanoparticlescomprising albumin and a substantially water insoluble pharmacologicallyactive agent, said method comprising subjecting a mixture comprising analbumin solution and an organic phase containing said substantiallywater insoluble pharmacologically active agent dispersed in an organicsolvent to a high shear condition, wherein the albumin was obtained by amethod comprising removing a prion protein from an initial albumincomposition. In some embodiments, there is provided a method ofproducing a composition comprising nanoparticles comprising albumin anda substantially water insoluble pharmacologically active agent, saidmethod comprising subjecting a mixture comprising an albumin solutionand an organic phase containing said substantially water insolublepharmacologically active agent dispersed in an organic solvent to a highshear condition, wherein the albumin was obtained by a method comprisingcontacting an initial albumin composition with a ligand capable ofbinding to a prion protein. In some embodiments, there is provided amethod of producing a composition comprising nanoparticles comprisingalbumin and a substantially water insoluble pharmacologically activeagent, said method comprising subjecting a mixture comprising an albuminsolution and an organic phase containing said substantially waterinsoluble pharmacologically active agent dispersed in an organic solventto a high shear condition, wherein the albumin was obtained by a methodcomprising: a) contacting an initial albumin composition with a ligandcapable of binding to a prion protein, and b) removing the ligand andprotein bound thereto from the initial composition.

In some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, saidmethod comprising: a) removing a prion protein from an initial albumincomposition; b) subjecting a mixture comprising a solution comprisingthe prion-removed albumin and an organic phase comprising saidsubstantially water insoluble pharmacologically active agent dispersedin an organic solvent to a high shear condition. In some embodiments,there is provided a method of producing a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, said method comprising: a) contacting aninitial albumin composition with a ligand capable of binding to a prionprotein to cause formation of a complex between the ligand and a prionprotein, b) removing the complex from the albumin initial composition;and c) subjecting a mixture comprising a solution comprising theprion-removed albumin and an organic phase comprising said substantiallywater insoluble pharmacologically active agent dispersed in an organicsolvent to a high shear condition.

In some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent,comprising: a) contacting an albumin solution with a ligand capable ofbinding to a prion protein, b) removing the ligand and proteins boundthereto from the albumin solution, and c) subjecting a mixturecomprising said albumin solution and an organic phase comprising saidsubstantially water insoluble pharmacologically active agent dispersedin an organic solvent to a high shear condition. In some embodiments,the mixture contains substantially no surfactants.

The prions can be removed during the formation of the nanoparticles. Forexample, in some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent,comprising: a) contacting a mixture comprising an albumin solution andan organic phase comprising said substantially water insolublepharmacologically active agent dispersed in an organic solvent with aligand capable of binding to a prion protein. In some embodiments, themethod further comprises: b) removing the ligand and proteins boundthereto from the mixture. In some embodiments, the method furthercomprises c) subjecting the mixture to a high shear condition. In someembodiments, the mixture contains substantially no surfactants.

The prion proteins can also be removed after the formation of thenanoparticle composition. For example, in some embodiments, there isprovided a method of producing a composition comprising nanoparticlescomprising albumin and a substantially water insoluble pharmacologicallyactive agent, comprising contacting a mixture comprising an organicphase comprising said substantially water insoluble pharmacologicallyactive agent dispersed in an organic solvent and an albumin solutionwith a ligand capable of binding to a prion protein, wherein the mixturehas been subjected to a high shear condition prior to contacting withthe ligand. In some embodiments, there is provided a method of producinga composition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent,comprising: a) subjecting a mixture comprising an organic phasecomprising said substantially water insoluble pharmacologically activeagent dispersed in an organic solvent and an albumin solution to a highshear condition, and b) contacting the mixture with a ligand capable ofbinding to a prion protein. In some embodiments, the method furthercomprises: c) removing the ligand and proteins bound thereto from themixture. In some embodiments, the mixture is substantially free ofsurfactants.

In some embodiments, there is provided a method of removing a prionprotein from a composition suspected of containing a prion proteincomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, said method comprising a)contacting the nanoparticle composition with a ligand capable of bindingto a prion protein, b) removing the ligand and proteins bound theretofrom the nanoparticle composition. In some embodiments, there isprovided a method of removing a prion protein from an albumincomposition suspected of containing an abnormal prion protein,comprising: a) contacting the composition comprising albumin with aligand capable of binding to a prion protein, b) removing the ligand andproteins bound thereto from the albumin composition, wherein saidalbumin composition is used to produce a composition comprisingnanoparticles comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent.

In some embodiments, there is provided a method of removing a prionprotein from a composition comprising nanoparticles comprising albuminand a substantially water insoluble pharmacologically active agent,comprising: a) determining the presence or absence of a prion protein inthe composition, b) contacting the composition with a ligand capable ofbinding to a prion protein, and c) removing the ligand and proteinsbound thereto from the composition.

Also provided are compositions made during the prion removal process.For example, in some embodiments, there is provided a compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, further comprising a ligandcapable of binding to a prion protein. In some embodiments, there isprovided a mixture comprising nanoparticle comprising albumin and asubstantially water insoluble pharmacologically active agent, and aligand capable of binding to a prion protein attached to a supportingmaterial, such as one or more supporting materials described herein. Insome embodiments, there is provided a column loaded with a compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, wherein the column comprises aligand capable of binding to a prion protein.

Also provided are compositions made by methods described herein. Alsoprovided are methods of using the prion-free compositions describedherein. For example, in some embodiments, there is provided a method ofadministering a composition comprising nanoparticles comprising albuminand a substantially water insoluble pharmacologically active agent,wherein the composition is substantially free of a prion protein. Insome embodiments, there is provided a method of treating a disease (suchas cancer) comprising administering a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, wherein the composition is substantiallyfree of a prion protein.

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe albumin in the composition was obtained by a method comprising aprion-removal process, said prion-removal process comprising contactingan initial albumin composition with a ligand capable of binding to aprion protein. In some embodiments, the prion removal process furthercomprises removing said ligand and proteins bound thereto from saidalbumin composition. In some embodiments, there is provided a method oftreating a disease (such as cancer) comprising administering acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe albumin in the composition was obtained by a method comprising aprion-removal process, said prion removal process comprising contactingan initial albumin composition with a ligand capable of binding to aprion protein.

Also provided are kits and dosage forms (such as vials for examplesealed vials) comprising the prion-free nanoparticle compositionsdescribed herein and kits useful for methods described herein. Furtherprovided are systems (including apparatus) for carrying out one or moremethods described herein.

These and other aspects and advantages of the present invention willbecome apparent from the subsequent detailed description and theappended claims. It is to be understood that one, some, or all of theproperties of the various embodiments described herein may be combinedto form other embodiments of the present invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides western blots and SDS-PAGE gels of DVR resin challengedwith 0.01% and 0.005% scrapie hamster brain homogenate in 20% or 25%albumin AMN31 was the positive control resin. The signal observed wasthe bound fraction. “−PK” and “+PK” denotes absence or presence ofProteinase K digestion.

FIG. 2 provides western blots and SDS-PAGE gels of YVHEA and SYA resinschallenged with 0.01% and 0.005% scrapie hamster brain homogenate in 20%or 25% albumin AMN31 was the positive control resin. The signal observedwas the bound fraction. “−PK” and “+PK” denotes absence or presence ofProteinase K digestion.

FIG. 3 provides western blots and SDS-PAGE gels of D4 resin challengedwith 0.01% and 0.005% scrapie hamster brain homogenate in 20% or 25%albumin AMN31 was the positive control resin. The signal observed wasthe bound fraction. “−PK” and “+PK” denotes absence or presence ofProteinase K digestion.

FIG. 4 depicts the process flow scheme for the TSE removal by the prionreduction resin column (PRDT (Pathogen Removal and DiagnosticTechnologies) column) for 20% albumin.

FIG. 5 shows a chromatography profile from the spiked run in the studyof TSE removal by the PRDT column for 20% albumin.

FIG. 6 shows the Western blot interference testing of 20% albuminsolution with centrifugation (with and without 10-fold concentration).

FIG. 7 depicts the process flow scheme for the TSE removal by the prionreduction resin column (PRDT column) for 25% albumin

FIG. 8 shows a chromatography profile from the spiked run in the studyof TSE removal by the PRDT column for 25% albumin.

FIG. 9 shows the Western blot interference testing of 25% albuminsolution with centrifugation (with and without 10-fold concentration).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides prion-free compositions (such aspharmaceutical compositions) comprising nanoparticles comprising albuminand substantially water insoluble pharmacologically active agents andmethods of making prion-free compositions.

The present invention in one aspect provides compositions (such aspharmaceutical compositions) comprising nanoparticles comprising albuminand substantially water insoluble pharmacologically active agents,wherein the composition is substantially free of a prion protein.

In another aspect, there is provided a method of making a prion-freecomposition (such as pharmaceutical compositions) comprisingnanoparticles comprising albumin and substantially water insolublepharmacologically active agent. Compositions made during the method ofmaking process are also provided.

In another aspect, there is provided a method of using a prion-freecomposition (such as pharmaceutical compositions) comprisingnanoparticles comprising albumin and substantially water insolublepharmacologically active agent.

Also provided are kits and dosage forms (such as vials for examplesealed vials) comprising the prion-free nanoparticle compositionsdescribed herein and kits and systems (including apparatus) useful formethods described herein.

“Prion free” is used herein for convenience and generally to describethe inventive compositions and is meant to encompass all embodimentsdescribed herein.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

It is understood that aspect and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of aspectsand embodiments.

Prion-Free Nanoparticle Compositions

The present invention provides a composition (such as a pharmaceuticalcomposition) comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe composition is substantially free of a prion protein. In someembodiments, the composition is sterile. In some embodiments, thecomposition is sterile filterable. In some embodiments, the compositionfurther comprises a pharmaceutically acceptable carrier.

In some embodiments, a prion protein cannot be detected in thecomposition by a detection method having a detection sensitivity ofabout 100 fg/ml or higher, that is, the test result is negative in anassay using a method having a detection sensitivity of about 100 fg/mlor higher (for example a detection sensitivity of about 50 fg/ml, about10 fg/ml, about 1 fg/ml, about 0.1 fg/ml). The prion content in thecomposition can be determined directly from the composition.Alternatively, the composition can be processed prior to thedetermination, for example, concentrated or enriched in order tofacilitate the detection and quantitation of the prion protein in thecomposition.

One way of determining whether or not a composition is substantiallyfree of a prion protein is in vivo infectivity assay. For example, invivo infectivity can be demonstrated by inoculation of the testingcomposition in mouse, mink, hamster, or goat models. Infectivity can bedetermined by lethal dose (LD50), i.e., the dose which when administeredby a given route (such as by intracerebral route) induces disease in 50%of exposed animals. Alternatively, infectivity can be determined byinfectious units, i.e., the minimal infectious dose capable oftransmitting the disease to one experimental animal to another by agiven route. Generally, 100 IU-ic/ml (infectious unit determined byintracerebral route) corresponds to about 10 LD50/ml and 1 pg/ml ofprion protein.

Another method for determining a prion protein in the composition isimmuno-polymerase chain reaction (IPCR), a technique whereby theexponential amplification ability of PCR is coupled to the detection ofproteins by antibodies in an ELISA format and is applied in a modifiedreal-time IPCR method to detect ultra-low levels of prion protein. SeeBarletta et al., J. Virology Method, 127 (2005):154-104. Using IPCR,recombinant hamster PrPc was consistently detected at 1 fg/ml andproteinase K (PK)-digested scrapie infected hamster brain homogenatesdiluted to 10⁻⁸ (approximately 10-100 infectious units) was detectedwith a semi-quantitative dose response.

In some embodiments, the prion protein in the composition is determinedby Protein Misfolding Cyclic Amplification (PMCA). This method has beenused to detect PrPsc in the blood. Other methods suitable fordetermining a prion protein in the composition include, but not limitedto: quantitative sandwich ELISA using time-resolveddissociation-enhanced fluorescence technology; dual-color fluorescentconfocal scanning; conformation dependent immunoassay (CDI). Westernblotting, bead blot, gel-mobility shift assays, fluorescent in situhybridization analysis (FISH), tracking of radioactive or bioluminescentmarkers, nuclear magnetic resonance, electron paramagnetic resonance,stopped-flow spectroscopy, column chromatography, capillaryelectrophoresis, or other methods can also be developed to detect prionproteins in the composition.

In some embodiments, the composition is substantially free of a prionprotein based on one of the assays for detecting prion proteins (such asany one of the assays described above). In some embodiments, two or moreassays are used to analyze the composition, and the correlation betweenthese different assays is used to determine whether or not thecomposition is free of a prion protein.

Thus, in some embodiments, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, wherein the composition is substantiallyfree of a prion protein. In some embodiments, the composition has aprion infectivity of less than about 100 fg/ml. In some embodiments, thecomposition has a prion infectivity of less than about 10 IU-ic/ml. Insome embodiments, the composition has a prion infectivity of less thanabout 1 LD50/ml. In some embodiments, the composition does not show thepresence of a prion protein based on the protein misfolding cyclicamplification (PMCA) assay. In some embodiments, the composition doesnot show the presence of a prion protein based on the IPCR assay. Insome embodiments, the composition has a prion infectivity of less thanabout 10 IU-ic/ml and does not show the presence of a prion proteinbased on the PMCA assay. In some embodiments, the In some embodiments,the composition has a prion infectivity of less than about 10 IU-ic/mland does not show the presence of a prion protein based on the IPRCassay. In some embodiments, the composition is free of prion proteinbased on the standard provided in the Guideline for the Investigation ofManufacturing Processes for Plasma-Derived Medicinal Products withRegard to vCJD Risk (CPMP5136/03), available athttp://www.emea.europa.eu/pdfs/human/bwp/513603en.pdf, the content ofwhich is incorporated herein in its entirety.

The compositions described herein are generally substantially free ofPrPsc. In some embodiments, the composition is also substantially freeof PrPc. In some embodiments, the molar ratio of PrPsc and PrPc in thecomposition is no greater than about 1:1, such as no great than aboutany one of 1:10, 1:100, 1:1000, 1:10000, or 1:100000.

In some embodiments, the composition is substantially free of a prionprotein from human, bovine, sheep, and rodent (such as hamster, mice,and mink) In some embodiments, the composition is substantially free ofthe H-type prion protein, the L-type prion protein, or both. In someembodiments, the composition is substantially free of cell-bound ofprion protein, the free prion protein, or both.

The term “PrPc” used herein refers to the native prion protein moleculewhich is naturally expressed within the body of the mammalian. The term“PrPsc” used herein refers to the conformationally altered form of thePrPc molecule that is thought to be infectious.

“Albumin” used herein refers to naturally occurring albumin and does notencompass albumin produced recombinantly. Naturally occurring albumin isadvantageous over recombinant albumin because it is the natural ligandfor albumin receptors in vivo. The albumin used in the methods describedherein generally retains the post-translational modifications of albuminand thus has reduced risks of immunogenicity. In some embodiments, thealbumin is obtained from a blood-derived composition. “Blood derivedcomposition” used herein include whole blood, red blood cellconcentrate, plasma, serum, platelet rich and platelet poor fraction,platelet concentrate, while blood cell, blood plasma precipitate, bloodplasma fractionation precipitate and supernatant, plasma fractionationintermediate, various other substances which are derived from blood, andthe like. In some embodiments, the albumin is from human. In someembodiments, the albumin is from an animal such as bovine, sheep, androdent (such as mouse, hamster, and mink) In some embodiments, thealbumin is obtained from a population of individuals (such as human) atleast some of which have been infected with prions. In some embodiments,the albumin is obtained from a population of individuals (such as human)at least some of which are suspected of having been infected withprions. In some embodiments, the albumin is obtained from a largebatch-size pool of individuals.

The composition described herein in some embodiments contains a traceamount of substances introduced during the prion-removal process. Forexample, in some embodiments, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, wherein the composition is substantiallyfree of a prion protein, and wherein the composition comprises a traceamount of a ligand capable of binding to a prion protein. In someembodiments, there is provided a composition comprising nanoparticlescomprising albumin and a substantially water insoluble pharmacologicallyactive agent, wherein the composition is substantially free of a prionprotein, and wherein the composition comprises a trace amount of asupporting material (such as material from a supporting materialdescribed herein, including a resin). In some embodiments, there isprovided a composition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe composition is substantially free of a prion protein, and whereinthe composition comprises a trace amount of a ligand capable of bindingto a prion protein and a trace amount of a supporting material (such asmaterial from a supporting material described herein, including aresin).

“Trace amount” refers to a detectable amount that does not affect theproperty of the composition, for example in terms of bioavailabilityand/or bioequivalency.

In some embodiments, the composition is bioequivalent to a compositionwherein the albumin has not been cleared by a prion-removal process.Bioequivalence can be established, for example, by a 90% confidenceinterval of between 0.80 and 1.25 for both Cmax and AUC, or a 90%confidence interval of between 0.80 and 1.25 of AUC and a 90% confidenceinterval of between 0.70 and 1.43 for Cmax.

In some embodiments, the level of an albumin stabilizer in thecomposition is less than that of a composition wherein the albumin hasnot been cleared by a prion-removal process. These albumin stabilizersinclude, for example, N-acetyl tryptophanate and sodium caprylate.

The compositions described herein generally encompass nanoparticlescomprising a substantially water insoluble pharmaceutically active agentand an albumin In some embodiments, the nanoparticle compositioncomprises nanoparticles comprising a substantially water insolublepharmacologically active agent and an albumin In some embodiments, thenanoparticles in the composition described herein have an averagediameter of no greater than about 1000 nm, including for example nogreater than about any one of 900, 800, 700, 600, 500, 400, 300, 200,190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 nm.In some embodiments, at least about 50% (for example at least about anyone of 60%, 70%, 80%, 90%, 95%, or 99%) of all the nanoparticles in thecomposition have a diameter of no greater than about 1000 nm, includingfor example no greater than about any one of 900, 800, 700, 600, 500,400, 300, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80,70, or 60 nm. In some embodiments, at least about 50% (for example atleast any one of 60%, 70%, 80%, 90%, 95%, or 99%) of all thenanoparticles in the composition fall within the range of about 20 toabout 200 nm, including for example any one of about 30 to about 180 nm,and any one of about 40 to about 150, about 50 to about 120, and about60 to about 100 nm.

In some embodiments, at least about 5% (including for example at leastabout any one of 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or90%) of the albumin in the nanoparticle portion of the composition arecrosslinked (for example crosslinked through one or more disulfidebonds).

In some embodiments, the nanoparticles comprise the substantially waterinsoluble pharmacologically active agent (such as paclitaxel) coatedwith an albumin, (e.g., human serum albumin). In some embodiments, thecomposition comprises substantially water insoluble pharmacologicallyactive agent in both nanoparticle and non-nanoparticle forms, wherein atleast about any one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of thesubstantially water insoluble pharmacologically active agent in thecomposition are in nanoparticle form. In some embodiments, thesubstantially water insoluble pharmacologically active agent in thenanoparticles constitutes more than about any one of 50%, 60%, 70%, 80%,90%, 95%, or 99% of the nanoparticles by weight. In some embodiments,the nanoparticles have a non-polymeric matrix. In some embodiments, thenanoparticles comprise a core of substantially water insolublepharmacologically active agent that is substantially free of polymericmaterials (such as polymeric matrix).

In some embodiments, the nanoparticle composition is substantially free(such as free) of surfactants or organic solvent (such as Cremophor®,Tween 80, or any other organic solvents used for the administration ofsubstantially water insoluble pharmacologically active agents). In someembodiments, the nanoparticle composition contains less than about anyone of 20%, 15%, 10%, 7.5%, 5%, 2.5%, 1% or less organic solvent.

The removal of prion from the albumin-containing composition makes itpossible to administer higher amounts of albumin without being concernedabout prions. The present invention thus also contemplates compositions(such as pharmaceutical compositions) comprising nanoparticlescomprising albumin and substantially water insoluble pharmacologicallyactive agent, wherein the weight ratio of albumin to the substantiallywater insoluble pharmaceutical agent is about 20:1 or more, such asabout any of about 30:1 or more, about 40:1 or more, or about 50:1 ormore. Exemplary ratios include, for example, about 20:1 to about 40:1,about 40:1 to about 60:1, about 60:1 to about 80:1, or about 90:1 toabout 100:1. In some embodiments, the weight ratio of albumin andsubstantially water insoluble pharmacologically active agent in thenanoparticle composition is about 18:1 or less, such as about 15:1 orless, for example about 10:1 or less. In some embodiments, the weightratio of albumin and substantially water insoluble pharmacologicallyactive agent in the composition falls within the range of any one ofabout 1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 to about13:1, about 4:1 to about 12:1, about 5:1 to about 10:1. In someembodiments, the weight ratio of albumin and substantially waterinsoluble pharmacologically active agent in the nanoparticle portion ofthe composition is about any one of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, or less.

In some embodiments, the particle composition comprises one or more ofthe above characteristics.

In some embodiments, the nanoparticle composition is Abraxane™.Nanoparticle compositions comprising other substantially water insolublepharmacologically active agents (such as docetaxel and ortataxel) mayalso comprise one or more of the above characteristics.

In some embodiments, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, wherein the albumin in the compositionwas obtained by a method comprising a prion-removal process, said prionremoval process comprising contacting an initial albumin compositionwith a ligand capable of binding to a prion protein. In someembodiments, the prion-removal process further comprises removing saidligand and proteins bound thereto from said albumin composition.

In some embodiments, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, wherein the albumin in the compositionwas obtained by a method comprising: a) contacting an initialcomposition comprising albumin with a ligand capable of binding to aprion protein to cause formation of a complex between the ligand and aprion protein, and b) removing the complex from the initial composition.

Nanoparticles comprising albumins and substantially water insolubledrugs are further described below in more detail. The method of removingprions from a composition (such as an initial albumin composition, ananoparticle composition comprising albumin and a substantially waterinsoluble pharmacologically active agent, or an intermediate compositionformed during the process of making the nanoparticles) are furtherdescribed below in more detail. The present invention encompassescompositions produced by any of the methods described herein.

The compositions described herein generally have reduced prion proteinlevel as compared to compositions wherein the albumin has not beencleared by a prion-removal process. For example, in some embodiments,the composition has less than about any of 50%, 40%, 30%, 20%, 10%, 5%,4%, 3%, 2%, 1%, or less of prion protein than a composition wherein thealbumin has not been cleared by a prion-removal process. In someembodiments, the composition has any of about 1, 2, 2.5, 3, 3.5, 4, 4.5,5, 6, 7, or 8 logs less prion protein than a composition wherein thealbumin has not been cleared by a prion-removal process. In someembodiments, the composition has any of about 1, 2, 2.5, 3, 3.5, 4, 4.5,5, 6, 7, or 8 logs less infectivity than a composition wherein thealbumin has not been cleared by a prion-removal process. In someembodiments, the composition of the present invention is bioequivalentto a composition wherein the albumin has not been cleared by aprion-removal process.

Although the present application focuses on albumin, it is to beunderstood that other proteins normally found in blood or plasma, whichinclude, but are not limited to, immunoglobulin (including IgA and IgG),lipoproteins, apolipoprotein B, alpha-acid glycoprotein,beta-2-macroglobulin, thyroglobulin, transferin, fibronectin, factorVII, factor VIII, factor IX, factor X, and the like, are alsocontemplated. All relevant descriptions about albumin provided hereinare equally applicable to these other proteins to the extent they areutilized in the formation of nanoparticles.

Methods of Making Prion-Free Nanoparticle Compositions

In another aspect, there are provided methods of producing prion-freenanoparticle compositions. For example, in some embodiments, there isprovided a method of producing a composition comprising nanoparticlescomprising albumin and a substantially water insoluble pharmacologicallyactive agent, said method comprising subjecting a mixture comprising analbumin solution and an organic phase containing said substantiallywater insoluble pharmacologically active agent dispersed in an organicsolvent to a high shear condition, wherein the albumin was obtained by amethod comprising removing a prion protein from an initial albumincomposition. In some embodiments, there is provided a method ofproducing a composition comprising nanoparticles comprising albumin anda substantially water insoluble pharmacologically active agent, saidmethod comprising subjecting a mixture comprising an albumin solutionand an organic phase containing said substantially water insolublepharmacologically active agent dispersed in an organic solvent to a highshear condition, wherein the albumin was obtained by a method comprisingcontacting an initial albumin composition with a ligand capable ofbinding to a prion protein. In some embodiments, there is provided amethod of producing a composition comprising nanoparticles comprisingalbumin and a substantially water insoluble pharmacologically activeagent, said method comprising subjecting a mixture comprising an albuminsolution and an organic phase containing said substantially waterinsoluble pharmacologically active agent dispersed in an organic solventto a high shear condition, wherein the albumin was obtained by a methodcomprising: a) contacting an initial albumin composition with a ligandcapable of binding to a prion protein, and b) removing the ligand andprotein bound thereto from the initial composition.

In some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, saidmethod comprising: a) removing a prion protein from an initial albumincomposition; b) subjecting a mixture comprising a solution comprisingthe prion-removed albumin and an organic phase comprising saidsubstantially water insoluble pharmacologically active agent dispersedin an organic solvent to a high shear condition. In some embodiments,there is provided a method of producing a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, said method comprising: a) contacting aninitial albumin composition with a ligand capable of binding to a prionprotein to cause formation of a complex between the ligand and a prionprotein, and b) removing the complex from the albumin initialcomposition; c) subjecting a mixture comprising a solution comprisingthe prion-removed albumin and an organic phase comprising saidsubstantially water insoluble pharmacologically active agent dispersedin an organic solvent to a high shear condition.

In some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent,comprising: a) contacting an albumin solution with a ligand capable ofbinding to a prion protein, b) removing the ligand and proteins boundthereto from the albumin solution, c) subjecting a mixture comprisingsaid albumin solution and an organic phase comprising said substantiallywater insoluble pharmacologically active agent dispersed in an organicsolvent to a high shear condition. In some embodiments, the mixturecontains substantially no surfactants.

The prions can be removed during the formation of the nanoparticles. Forexample, in some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent,comprising: a) contacting a mixture comprising an albumin solution andan organic phase comprising said substantially water insolublepharmacologically active agent dispersed in an organic solvent with aligand capable of binding to a prion protein. In some embodiments, themethod further comprises: b) removing the ligand and proteins boundthereto from the mixture. In some embodiments, the method furthercomprises c) subjecting the mixture to a high shear condition. In someembodiments, the method further comprises removing the organic solventfrom the mixture. In some embodiments, the mixture containssubstantially no surfactants.

In some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent,comprising: a) subjecting a mixture comprising an albumin solution andan organic phase comprising a substantially water insolublepharmacologically active agent dispersed in an organic solvent to a highshear condition; and b) contacting the mixture with a ligand capable ofbinding to a prion protein. In some embodiments, the method furthercomprises: c) removing the ligand and proteins bound thereto from themixture. In some embodiments, the method further comprises removing theorganic solvent from the mixture. In some embodiments, the mixturecontains substantially no surfactants.

In some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, comprisingcontacting a mixture comprising an organic phase comprising saidsubstantially water insoluble pharmacologically active agent dispersedin an organic solvent and an albumin solution with a ligand capable ofbinding to a prion protein, wherein the mixture has been subjected to ahigh shear condition prior to contacting with the ligand. In someembodiments, there is provided a method of producing a compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, comprising: a) subjecting amixture comprising an organic phase comprising said substantially waterinsoluble pharmacologically active agent dispersed in an organic solventand an albumin solution to a high shear condition, and b) contacting themixture with a ligand capable of binding to a prion protein. In someembodiments, the method further comprises: c) removing the ligand andproteins bound thereto from the mixture. In some embodiments, the methodfurther comprises: d) removing the aqueous phase from the mixture. Insome embodiments, the mixture is substantially free of surfactants.

In some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent,comprising: a) subjecting a mixture comprising an organic phasecomprising said substantially water insoluble pharmacologically activeagent dispersed in an organic solvent and an albumin solution to a highshear condition, b) removing said organic solvent, and c) contacting themixture with organic solvent removed with a ligand capable of binding toa prion protein. In some embodiments, the method further comprises: d)removing the ligand and proteins bound thereto from the mixture. In someembodiments, the method further comprises: d) removing the aqueous phasefrom the mixture. In some embodiments, the mixture is substantially freeof surfactants.

In some embodiments, there is provided a method of producing acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent,comprising: a) subjecting a mixture comprising an organic phasecomprising said substantially water insoluble pharmacologically activeagent dispersed in an organic solvent and an albumin solution to a highshear condition, b) removing said organic solvent, c) adding albumin tothe mixture, and d) contacting the mixture with a ligand capable ofbinding to a prion protein. In some embodiments, the method furthercomprises: e) removing the ligand and proteins bound thereto from themixture. In some embodiments, the method further comprises: f) removingthe aqueous phase from the mixture. In some embodiments, the mixture issubstantially free of surfactants.

The methods described herein generally include the step of subjecting amixture comprising an organic phase comprising the substantially waterinsoluble pharmacologically active agent dispersed in an organic solventand an albumin solution to a high shear condition. In some embodiments,the high shear condition is high pressure homogenization, for example ata pressure in the range of about 3000 to about 30,000 psi, including forexample about 6000 to about 25,000 psi, about 9000 to about 18,000 psi,about 10,000 to about 25,000 psi, about 15,000 to about 25,000 psi. Insome embodiments, organic solvent is a mixture of a substantially waterimmiscible organic solvent (such as chloroform or methylene chloride)and a water soluble organic solvent (such as a water soluble alcohol,including ethanol and t-butanol). In some embodiments, the ratio (v/v)of the substantially water immiscible organic solvent and the watersoluble organic solvent (for example the ratio of chloroform/ethanol orchloroform/butanol) is about any of 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3,1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1, or with a ratio ofabout any of 3:7, 5:7, 4:6. 6:4, 5:5, 6:5, 8:5, 9:5, 9.5:5, 5:3, 7:3,6:4, or 9.5:0.5.

In some embodiments, the method further comprises removing the organicphase from the mixture (such as removal by evaporation under reducedpressure). In some embodiments, the method further comprises removingthe aqueous phase from the mixture. In some embodiments, the methodfurther comprises sterile filtering the nanoparticles formed by themethod described above.

In some embodiments, there is provided a method of removing a prionprotein from a composition suspected of containing a prion proteincomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, said method comprising: a)contacting the nanoparticle composition with a ligand capable of bindingto a prion protein, b) removing the ligand and proteins bound theretofrom the nanoparticle composition. In some embodiments, there isprovided a method of removing a prion protein from an albumincomposition suspected of containing an abnormal prion protein,comprising: a) contacting the composition comprising albumin with aligand capable of binding to a prion protein, b) removing the ligand andproteins bound thereto from the albumin composition, wherein saidalbumin composition is used to produce a composition comprisingnanoparticles comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent.

In some embodiments, there is provided a method of removing a prionprotein from a composition comprising nanoparticles comprising albuminand a substantially water insoluble pharmacologically active agent,comprising: a) determining the presence or absence of a prion protein inthe composition, b) contacting the composition with a ligand capable ofbinding to a prion protein, and c) removing the ligand and proteinsbound thereto from the composition.

In some embodiments, one or more steps of the methods described hereinare carried out in batch mode. In some embodiments, one of more steps ofthe methods described herein are carried out in continuous mode.

Removing Prion Prior to the Formation of Nanoparticles

The prion protein can be removed from an initial albumin compositionbefore the albumin-containing nanoparticle compositions are made.Generally, the method comprises contacting an initial albumincomposition with a ligand capable of binding to a prion protein, andremoving the ligand and protein bound thereto from the albumincomposition. This process can be repeated one or more times, with thesame or a different ligand. Two or more ligands can also be usedsimultaneously during the prion removal process.

In some embodiments, the initial albumin composition is a blood derivedcomposition. For example, in some embodiments, the initial albumincomposition is whole blood, red blood cell concentrate, plasma, serum,platelet rich and platelet poor fraction, platelet concentrate, whileblood cell, blood plasma precipitate, blood plasma fractionationprecipitate and supernatant, or plasma fractionation intermediate. Insome embodiments, the initial albumin composition is obtained fromhuman. In some embodiments, the initial albumin composition is from ananimal such as bovine, sheep, and rodent (such as mouse, hamster, andmink) In some embodiments, the initial albumin composition is obtainedfrom a population of individuals (such as human) at least some of whichhave been infected with prions. In some embodiments, the initial albumincomposition is obtained from a population of individuals (such as human)at least some of which are suspected of having been infected withprions.

In some embodiments, the initial albumin composition is an albumincomposition prepared from a body fluid (such as blood) by any of variousmethods common in the art including ion exchange, affinity, gelpermeation, and/or hydrophobic chromatography and/or by differentialprecipitation. In some embodiments, the initial composition is analbumin composition purified from the blood (such as human blood). Insome embodiments, the initial albumin composition is an albumincomposition purified from serum (such as human serum). In someembodiments, the initial albumin composition has a prion infectivity ofabout 100 IU-ic/ml, 90IU-ic/ml, 50IU-ic/ml, or 10IU-ic/ml. In someembodiments, the concentration of albumin in the initial albumincomposition is about 1% (w/v), including for example about 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30%.

During the prion-removal process, the ligand is brought into contactwith the initial albumin composition and allowed to bind to prionproteins in the initial albumin composition. Conditions suitable for thebinding can be determined and optimized to facilitate binding of theligand to a prion protein based on the nature of the ligand and itsbinding specificity to the prion protein. The binding in someembodiments is carried out at a temperature of about 0° C. to about 39°C., including for example about 20° C. to about 25° C. The binding canbe carried out at pH of about 4 to about 10, including for example about5 to about 9, about 6 to about 8, about 6.8 to about 7.5, about 6.9 toabout 7.4, or about 7. Optionally, blocking agents can be used to reducenon-specific binding to the ligand.

After the contacting step, the ligand and proteins bound thereto areremoved from the rest of the composition. The term “removing” as usedherein refers to the separation of the ligand and protein bound theretofrom the albumin-containing composition. The separation can be carriedout in a variety of ways, depending on the nature of the ligand and thesupporting material (if any) used to facilitate the separation. Forexample, the ligand and proteins bound thereto can be separated out bychromatography, such as, but not limited to, thin-layer, column andbatch chromatography; solid support and membrane separation; reactorseparation; magnetic separation, immunoseparation; colloidal separation;sedimentation; precipitation; or centrifugation.

In some embodiments, ligand may be attached to a supporting materialsuch as a bead or a membrane, which in turn is allowed to contact theinitial albumin composition. Ligand-immobilized support is allowed tocontact the initial albumin composition under a condition sufficient tocause formation of a prion-ligand complex. The solid phase is thenseparated from the composition, thereby removing the prion protein boundto the ligand from the sample. For example, in one exemplary embodiment,the ligands are immobilized in a column, such as a chromatographycolumn, a sample (such as the initial albumin composition) is thenpassed through the column either due to the force of gravity or underpressure, such as in a high pressure liquid chromatography column. Prionproteins in the sample will bind to the ligand immobilized on thecolumn, and the sample passing through can be collected. This processcan repeat several times to achieve the desired result, using the sameor different ligands.

The flow rate of a sample (such as the initial albumin composition) in acolumn can be adjusted to maximize the binding of the ligand and theprion proteins in a sample. In some embodiments, the binding is carriedout at a flow rate of about 0.1 ml per minute to about 5.0 ml perminute, about 0.1 ml per minute to about 2.5 ml per minute, about 0.1 mlper minute to about 0.25 ml per minute, about 0.25 per minute to about0.5 ml per minute, about 0.5 ml per minute to about 1.0 ml per minute,about 1.0 ml per minute to about 1.5 ml per minute, about 1.5 ml perminute to about 2.0 ml per minute, about 2.0 ml per minute to about 2.5ml per minute, about 2.5 ml per minute to about 3.0 ml per minute, about3.0 ml per minute to about 3.5 ml per minute, about 3.5 ml per minute toabout 4.0 ml per minute, about 4.0 ml per minute to about 4.5 ml perminute, or about 4.5 ml per minute to about 5.0 ml per minute, includingfor example about 0.1 ml per minute, 0.25 ml per minute, 0.5 ml perminute, 1.0 ml per minute, 1.5 ml per minute, 1.7 ml per minute, 1.8 mlper minute, 1.9 ml per minute, 2.0 ml per minute, 2.1 ml per minute, 2.3ml per minute, 2.5 ml per minute, 2.7 ml per minute, 3.0 ml per minute,3.5 ml per minute, 4.0 ml per minute, 4.5 ml per minute, or 5.0 ml perminute. In some embodiments, the flow rate is at least about 10 ml perminute, such as at least about any of 20 ml per minute, 30 ml perminute, 40 ml per minute, 50 ml per minute.

The total flow-through volume or total flow-through time during abinding process can also be adjusted to maximize the binding of theligand and the prion proteins in a sample (such as the initial albumincomposition). In some embodiments, the total flow-through volume isabout 1 time to about 1000 times of the column volume, including forexample about 2 times to about 10 times of the column volume, about 10times to about 20 times of the column volume, about 20 times to about 30times of the column volume, about 30 times to about 40 times of thecolumn volume, about 40 times to about 50 times of the column volume,about 50 times to about 1000 times of the column volume, about 50 timesto about 500 times of the column volume, about 100 times to about 600times of the column volume, or about 200 times to about 800 times of thecolumn volume. In some embodiments, the total flow-through volume isabout 100 times of the column volume. In other embodiments, the totalflow-through volume is about 500 times of the column volume. In someembodiments, the total flow-through time is about 1 hour to about 30hours, including for example about 2 hours to about 25 hours, about 3hours to about 20 hours, or about 3 hours to about 17 hours. In someembodiments, the total flow-through time is any of about 3 hours, 4hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 17hours, 18 hours, 19 hours, or 20 hours. In some embodiments, the totalflow through time is more than about 24 hours. In some embodiments, thetotal flow through time is less than about 8, including for example anyof 7, 6, 5, 4, 3, 2, 1, or 0.5 hours.

Alternatively, the ligand can first be brought into contact with theinitial albumin composition, under a condition sufficient to causeformation of a prion-ligand complex. The prion-ligand complex is thensubsequently removed by using a column, such as an affinity-basedchromatography. To facilitate the separation, the ligand may beconjugated to a binding partner so that the ligand/prion complex can beremoved via the binding partner, for example by using an affinity columncontaining a molecule that recognizes the binding partner.

In addition to batch or column chromatography, a variety of otherconfigurations, modifications and variations of the use of the ligandsfor binding prion proteins are also envisioned. Such variations andmodifications include, but are not limited to: batch processes,continuous processes, moving bed chromatography processes; low, medium,or high pressure processes; or small, medium or large scale processes.In some embodiments, the ligands are on a membrane, fibers bead,impregnated into a non-woven mesh, or coating fibers contained within afilter housing.

In some embodiments, the removal step does not significantly result inyield loss and/or change in the property and/or stability of the albuminIn some embodiments, the recovery of the albumin in its originalbiological state is substantially maintained at least to a level inexcess of 50%, including for example 80%, or 90%, or more. In someembodiments, the recovery rate of albumin from the prion removal processis higher than any of about 80%, 90%, 95%, or 99%. In some embodiments,the concentration of albumin in the initial albumin composition isadjusted or controlled prior to the prion removal step in order tominimize non-specific binding and loss of albumin during the process.For example, the concentration of albumin can be in the range of about1% to about 50%, about 5% to about 25%, about 5% to about 30%, about 5%to about 40%, about 5% to about 10%, about 10% to about 15%, about 15%to about 20%, about 20% to about 25%, about 25% to about 30% etc.,including for example about 5%, 10%, 15%, 20%, 25%, or 30% albumin

The resulting albumin composition can be analyzed to determine theclearance rate of the prion removal process. The ligand with bound prionproteins may also be analyzed (directly or after elution) to determinethe clearance rate.

The removal of prion proteins can be evaluated based on reduction ofprion protein or reduction of infectivity. In some embodiments, at leastabout 50%, including for example at least about 60%, 70%, 80%, 90%, 95%,99%, or 100% of the prion proteins are removed from the initial albumincomposition. In some embodiments, the infectivity of the post-removalalbumin composition is at least about 10×, 20×, 30×, 40×, 50×, 80×,100×, 200×, 500×, 1000×, 10⁴×, 10⁵×, 10⁶×, 10⁷×, 10⁸×, 10⁹× less thanthat of the initial albumin composition.

In some embodiments, serial infectivity is used to determine theclearance rate of the prion removal process. Serial dilutions of asamples are made and dilutions are examined for infectious activity, forexample in an assay animal. The dilution at which half of the animalsbecome infected is the infectious titer. For example, if a 5 folddilution is required, the sample may be defined as having 5 logs ofinfectivity. By comparing the log infectivity of the initial albumincomposition and that of the post-removal albumin composition, one candetermine the clearance rate of the prion removal process. In someembodiments, the prion removal method results in a reduction of any oneof 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, or 4, 5, 6, 7,8, 9, or 10 logs of infectivity.

In some embodiments, the clearance rate of the prion-removal process isdetermined based on spiking experiments with infectious materials byfollowing steps described herein for the prion-removal method. Suitablespiking agents include, but are not limited to, brain homogenates,microsomes, caveolae-like domains, purified PrPsc, and prion fibrils. Insome embodiments, the spiking agent is detergent solublized (such assarkosyl solubilized). In some embodiments, the spiking ratio in thecomposition is in the range of about 0.001% to about 5%, about 0.001% toabout 0.25%, about 0.001% to about 0.1%, about 0.001% to about 0.005%,about 0.005% to about 0.075%, about 0.075% to about 0.01%, about 0.01%to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 0.75%,about 0.75% to about 1%, about 1% to about 2%, about 2% to about 3%, orabout 3% to about 5%, including for example about 0.001%, 0.005%,0.075%, 0.01%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, or 5%.

In some embodiments, the reduction factors (RF) is used to determine theclearance rate of the prion removal process. The RF can be calculatedusing the formula:

RF=(V ₁ ×T ₁)/(V ₂ ×T ₂)

or

Log₁₀[RF]=[ Log₁₀(V ₁)+Log₁₀(T ₁)]−[ Log₁₀(V ₂)+Log₁₀((T ₂)].

Wherein V₁ and T₁ are the volume and titre of the initial albumincomposition, respectively, and V₂ and T₂ are the volume and titre of thepost-removal albumin composition. Reduction factors can be rounded to 1decimal place after the final calculation. In some embodiments, areduction factor of at least about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 log₁₀) infectivity of theprion proteins is removed from the initial albumin composition. Forexample, the prion protein can be removed by a reduction factor ofgreater than or equal to 2.5 log₁₀ in a 0.5% sarkosyl solubilizedfraction spiked into a 20% albumin composition. As another example, theprion protein can also be removed by a reduction factor of greater thanor equal to 2.0 log₁₀ in a 0.5% sarkosyl solubilized fraction spikedinto a 25% albumin composition.

The removal of prions can be evaluated by standard Western blotanalysis. For example, the post-binding ligands can first be treatedwith Proteinase K, which digests all PrPc but not PrPsc. The digest isthen run on SDS gel and transblotted to a sheet of nitrocellulose orPVDF membrane. The separated PrPsc bands are then visualized using 3F4or 6H4. 3F4 reacts with amino acid residues 109-112 PrP from humans,hamsters, and felines. In one exemplary embodiment, incubation wascarried out at a concentration of 0.6 ug/ml for a minimum of one hour,after which excess antibody was washed away and the membranes incubatedwith a rabbit anti-mouse horse-radish peroxidase conjugate (1:1000dilution) for a minimum of one hour. After extensive washing with TTBS,the membranes were developed using enhanced chemiluminescence. In someembodiments, the removal of prion proteins is evaluated according to theGuideline for the Investigation of Manufacturing Processes forPlasma-Derived Medicinal Products with Regard to vCJD Risk(CPMP5136/03).

Ligands Capable of Binding to a Prion Protein and Supporting Material

“Ligand” used herein refers to a molecule to which a prion protein orpeptide binds. A “ligand capable of binding to a prion protein” refersto a ligand that specifically binds to a prion protein under suitableconditions. In some embodiments, the ligand specifically binds to ahuman prion protein. In some embodiments, the ligand specifically bindsto a hamster prion protein. In some embodiments, the ligand specificallybinds to a mouse protein. In some embodiments, the ligand has binds toprion proteins from multiple species. For example, in some embodiments,the ligand binds to human prion protein, hamster prion protein, andmouse prion protein.

In some embodiments, the ligand binds to the prion protein (such as ahuman prion protein, hamster prion protein, and/or mouse prion protein)with a high binding affinity. For example, in some embodiments, theligand has a binding Kassociation of more than about any of 10⁷, 10⁸,10⁹, 10¹⁰, or 10¹¹.

A number of ligands have been identified that bind to prion protein andthus can be used in methods of the present invention. See, e.g.,WO04/090102, WO04/050851, WO06/010915, and WO06/044459. These include,for example, peptides, chemical compounds, and antibodies thatspecifically recognize a prion protein. The ligand can be used to removeall forms of prion proteins from a composition or can be selectivelychosen to detect or remove a single form of prion protein.

In some embodiments, the ligand for removing prion is a peptide, such aspeptides described in PCT published application No. WO04/05051. Forexample, in some embodiments, the ligand is a peptide having an aminoacid of any of SEQ ID NOs: 1-232 as shown in Table 1. In someembodiments, the ligand is a tripeptide, such as peptides having anamino acid sequence of any one of SEQ ID NO:48, 102, 105, 108, 109, 110,111, 143, 148, 193, 194, 195, 202, 203, 204, or 210. In someembodiments, the ligand is a peptide with six amino acids, such as6-mers having an amino acid sequence of any one of SEQ ID NO: 152, 153,180, 181, 182, 183, 185, 186, 187, 188, 189, or 190. In someembodiments, the ligand has an amino acid sequence of SEQ ID NO:150 or151.

TABLE 1 Amino acid sequences binding to prion sequences KIHKFLA (SEQ IDNO: 1) GTHDFQA (SEQ ID NO: 2) KFGSTHA (SEQ ID NO: 3) FVNEIEA (SEQ ID NO:4) GLHFKSA (SEQ ID NO: 5) GRVLHHA (SEQ ID NO: 6) QKNSEWA (SEQ ID NO: 7)HAYFTHA (SEQ ID NO: 8) WPKGAVA (SEQ ID NO: 9) RPWKKAA (SEQ ID NO: 10)PKHIWPA (SEQ ID NO: 11) HKLWGVA (SEQ ID NO: 12) GGYKPYA (SEQ ID NO: 13)ENVSQNA (SEQ ID NO: 14) HTYYNGA (SEQ ID NO: 15) KKKSDHA (SEQ ID NO: 16)HHLKGTA (SEQ ID NO: 17) KKHGVWA (SEQ ID NO: 18) DGTQAHA (SEQ ID NO: 19)APHRNNA (SEQ ID NO: 20) HHGHNIA (SEQ ID NO: 21) HTWHGQA (SEQ ID NO: 22)HVFVTWA (SEQ ID NO: 23) THHFYIA (SEQ ID NO: 24) KLGWG(A/G)A (SEQ ID NO:25) GSKKKEA (SEQ ID NO: 26) PLLVVWA (SEQ ID NO: 27) WLLVGGA (SEQ ID NO:28) (W/G)QVLVYA (SEQ ID NO: 29) RRHQRQA (SEQ ID NO: 30) LPWTFGA (SEQ IDNO: 31) IFIIITA (SEQ ID NO: 32) P(X)IEPHA (SEQ ID NO: 33) EWGIIWA (SEQID NO: 34) GWYIYFA (SEQ ID NO: 35) TLILFHA (SEQ ID NO: 36) FLLSNHA (SEQID NO: 37) WQIRFFA (SEQ ID NO: 38) VLLVFEA (SEQ ID NO: 39) GWVLEIA (SEQID NO: 40) FLLIDTA (SEQ ID NO: 41) GFLFKFA (SEQ ID NO: 42) PWTIYIA (SEQID NO: 43) WH (SEQ ID NO: 44) WW (SEQ ID NO: 45) LW (SEQ ID NO: 46) WNA(SEQ ID NO: 47) EFW (SEQ ID NO: 48) LPW (SEQ ID NO: 49) YEY (SEQ ID NO:50) WPA (SEQ ID NO: 51) FNQ (SEQ ID NO: 52) YHE (SEQ ID NO: 53) LFA (SEQID NO: 54) NHY (SEQ ID NO: 55) TLG (SEQ ID NO: 56) WVD (SEQ ID NO: 57)YWDQA (SEQ ID NO: 58) YVHEA (SEQ ID NO: 59) WFDEA (SEQ ID NO: 60) LQWYDA(SEQ ID NO: 61) YTHSEA (SEQ ID NO: 62) WIDYEA (SEQ ID NO: 63) VWIDAA(SEQ ID NO: 64) WDEAEEA (SEQ ID NO: 65) YDSYDDA (SEQ ID NO: 66) NDFIDFA(SEQ ID NO: 67) YEPWGSA (SEQ ID NO: 68) EYGDWWA (SEQ ID NO: 69) WDYDQEA(SEQ ID NO: 70) DWGDPFA (SEQ ID NO: 71) DWPEVWA (SEQ ID NO: 72) FHDFSEA(SEQ ID NO: 73) DTFWDYA (SEQ ID NO: 74) WNDLDNA (SEQ ID NO: 75) ASALVYA(SEQ ID NO: 76) LINAGGA (SEQ ID NO: 77) WESYVTA (SEQ ID NO: 78) WSDEGYA(SEQ ID NO: 79) YRWTGPA (SEQ ID NO: 80) YEDQWQA (SEQ ID NO: 81) EWADDNA(SEQ ID NO: 82) YEIDYGA (SEQ ID NO: 83) EFGYFDA (SEQ ID NO: 84) WGDEQDA(SEQ ID NO: 85) HEEDWAA (SEQ ID NO: 86) FEDFELA (SEQ ID NO: 87) TWGIDEA(SEQ ID NO: 88) WDPTDYA (SEQ ID NO: 89) NDKIHTA (SEQ ID NO: 90) FEDFFSA(SEQ ID NO: 91) YEWAEQA (SEQ ID NO: 92) THVYFLA (SEQ ID NO: 93)(S/T/W)XDFSDA (SEQ ID NO: 94) YRTPNEA (SEQ ID NO: 95) (GIL)RSETA (SEQ IDNO: 96) IHN (SEQ ID NO: 97) WEY (SEQ ID NO: 98) DYW (SEQ ID NO: 99) WDW(SEQ ID NO: 100) WQD (SEQ ID NO: 101) YFE (SEQ ID NO: 102) NYE (SEQ IDNO: 103) SYA (SEQ ID NO: 104) WDL (SEQ ID NO: 105) WLE (SEQ ID NO: 106)VQR (SEQ ID NO: 107) YID (SEQ ID NO: 108) RWD (SEQ ID NO: 109) DVR (SEQID NO: 110) WSD (SEQ ID NO: 111) HWD (SEQ ID NO: 112) WQD (SEQ ID NO:113) WDD (SEQ ID NO: 114) WED (SEQ ID NO: 115) ITN (SEQ ID NO: 116) YED(SEQ ID NO: 117) VADEEA (SEQ ID NO: 118) YYVDAA (SEQ ID NO: 119) QDFNLA(SEQ ID NO: 120) DNPIDA (SEQ ID NO: 121) FNEHEA (SEQ ID NO: 122) WGADGA(SEQ ID NO: 123) VIYSHA (SEQ ID NO: 124) HILEEA (SEQ ID NO: 125) PHENFA(SEQ ID NO: 126) EDNGGA (SEQ ID NO: 127) DSEGPA (SEQ ID NO: 128) FQEFTA(SEQ ID NO: 129) EGDEIA (SEQ ID NO: 130) IYAETA (SEQ ID NO: 131) RVRETA(SEQ ID NO: 132) EEPQWA (SEQ ID NO: 133) EGEEFA (SEQ ID NO: 134)(T/L)FNIHA (SEQ ID NO: 135) YDW (SEQ ID NO: 136) NYT (SEQ ID NO: 137)SYT (SEQ ID NO: 138) WAD (SEQ ID NO: 139) QWG (SEQ ID NO: 140) WGD (SEQID NO: 141) EYF (SEQ ID NO: 142) WEH (SEQ ID NO: 143) LYD (SEQ ID NO:144) DYY (SEQ ID NO: 145) FYE (SEQ ID NO: 146) EYY (SEQ ID NO: 147) YDY(SEQ ID NO: 148) WDH (SEQ ID NO: 149) RES(na)NVA (SEQ ID NO: 150)ES(na)PRQA (SEQ ID NO: 151) VARENIA (SEQ ID NO: 152) RWEREDA (SEQ ID NO:153) EWWETV (SEQ ID NO: 154) SVYQLDA (SEQ ID NO: 155) (na)HEFYGA (SEQ IDNO: 156) HE(na)(na)LVA (SEQ ID NO: 157) A(na)VPV(na)A (SEQ ID NO: 158)YFDYWLA (SEQ ID NO: 159) FE(na)HRQA (SEQ ID NO: 160) WRHEPAA (SEQ ID NO:161) SS(na)KKDA (SEQ ID NO: 162) R(na)DKEAA (SEQ ID NO: 163) (na)HEIFPA(SEQ ID NO: 164) aKWYHHRA (SEQ ID NO: 165) HWWPHNA (SEQ ID NO: 166)HWQVFYA (SEQ ID NO: 167) FHE(na)EIA (SEQ ID NO: 168) HADF(na)QA (SEQ IDNO: 169) ALHFETA (SEQ ID NO: 170) DDPTGFA (SEQ ID NO: 171) VAPGLGA (SEQID NO: 172) IFRLIEA (SEQ ID NO: 173) GLERPEA (SEQ ID NO: 174) IVVRLWA(SEQ ID NO: 175) WHNPHYA (SEQ ID NO: 176) LIYKSDA (SEQ ID NO: 177)EKPIFNA (SEQ ID NO: 178) HWSEPAA (SEQ ID NO: 179) GHNWKEA (SEQ ID NO:180) YWHHDDA (SEQ ID NO: 181) GYPKENA (SEQ ID NO: 182) PVYWLYA (SEQ IDNO: 183) FGEHTPA (SEQ ID NO: 184) FQGTREA (SEQ ID NO: 185) TGTNRYA (SEQID NO: 186) KWATRYA (SEQ ID NO: 187) NSTKFDA (SEQ ID NO: 188) LIYKEEA(SEQ ID NO: 189) EHATYRA (SEQ ID NO: 190) HND (SEQ ID NO: 191) HER (SEQID NO: 192) HGD (SEQ ID NO: 193) HSD (SEQ ID NO: 194) HFD (SEQ ID NO:195) WND (SEQ ID NO: 196) YEH (SEQ ID NO: 197) HWD (SEQ ID NO: 198) YHD(SEQ ID NO: 199) YDW (SEQ ID NO: 200) WDY (SEQ ID NO: 201) HYD (SEQ IDNO: 202) HWD (SEQ ID NO: 203) WTD (SEQ ID NO: 204) FPK (SEQ ID NO: 205)HWK (SEQ ID NO: 206) WEE (SEQ ID NO: 207) LLR (SEQ ID NO: 208) SYF (SEQID NO: 209) EYY (SEQ ID NO: 210) DRDLTFA (SEQ ID NO: 211) HNWWIIA (SEQID NO: 212) EVKIGNA (SEQ ID NO: 213) SIV (SEQ ID NO: 214) AYP (SEQ IDNO: 215) EVADEEA (SEQ ID NO: 216) EYYVDAA (SEQ ID NO: 217) YDNPIDA (SEQID NO: 218) YFNEHEA (SEQ ID NO: 219) EWGADGA (SEQ ID NO: 220) DVIYSHA(SEQ ID NO: 221) WHILEEA (SEQ ID NO: 222) NPHENFA (SEQ ID NO: 223)HEDNGGA (SEQ ID NO: 224) SDSEGPA (SEQ ID NO: 225) EFQEFTA (SEQ ID NO:226) QEGDEIA (SEQ ID NO: 227) DIYAETA (SEQ ID NO: 228) DRVRETA (SEQ IDNO: 229) FEEPQWA (SEQ ID NO: 230) FEGEEFA (SEQ ID NO: 231) (T/L)FNIHA(SEQ ID NO: 232)

In some embodiments, the ligand is a peptide of the amino acid sequenceof DVR, SYA, AMN31, D4, or YVHEA. In some embodiments, the ligand is apeptide that binds to a prion protein at an affinity that is similar toor higher than that of DVR, SYA, AMN31, D4, or YVHEA. In someembodiments, the ligand is DVR. In other embodiments, the ligand isAMN31. In some embodiments, the peptide ligands are provided in the formof resins (such as bound to resins).

In some embodiments, the ligand is an antibody recognizing a prionprotein. Antibodies recognizing prion proteins are known in the art.These include, but are not limited to, monoclonal antibodies 3F4, 6H4,or 16A18. In some embodiments, the antibody is a glycoform specificantibody, such as ICSM-4 and ICSM-10. Suitable antibodies useful for themethods described herein include polyclonal and monoclonal antibodies,single chain antibodies, Fab fragments, and Fv fragments.

In some embodiments, the ligand binds to a specific sequence of a prionprotein. For example, in some embodiments, the ligand (such as a peptideligand or an antibody ligand) binds to any one of the prion proteinsequences SEQ ID NOs: 133-146 listed on Table 2.

TABLE 2 Prion amino acid sequences RYPxQ, x is G, P, or N (SEQ ID NO:233) xxYYux, x is G, P, or N, u is R (SEQ ID NO: 234) or Q RYPGQ (SEQ IDNO: 235) DRYYRD (SEQ ID NO: 236) QAYYQR (SEQ ID NO: 237) QVYYRP (SEQ IDNO: 238) PHGGGWGQ (SEQ ID NO: 239) PHGGSWGQ (SEQ ID NO: 240) PHGGGWSQ(SEQ ID NO: 241) PHGGGGWSQ (SEQ ID NO: 242) PHGGGSNWGQ (SEQ ID NO: 243)PHNPGY (SEQ ID NO: 244) PHNPSY (SEQ ID NO: 245) PHNPGY (SEQ ID NO: 246)

In some embodiments, the ligand is a chemical compound. Compoundscapable of binding to a prion protein can be found, for example, in PCTApplication publication No. WO06/010915, which is incorporated herein inits entirety. In some embodiments, the ligand is a substituted triazine.In some embodiments, the ligand is a compound having formula (I):

wherein R¹ and R² are the same or different and are each optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl or optionally substituted heteroaryl groups; R³ ishydrogen or an aryl group substituent or R³ is a solid supportoptionally attached via a spacer; Z represents an oxygen atom, a sulphuratom or NR⁴; Y represents an oxygen atom, a sulphur atom or NR⁵; inwhich R⁴ and R⁵, which may be the same or different, represent hydrogen,optionally substituted alkyl containing 1 to 6 carbon atoms, optionallysubstituted phenyl, optionally substituted benzyl or optionallysubstituted β-phenylethyl; and one of X¹ and X² represents a nitrogenatom and the other of X¹ and X² represents a nitrogen atom or CR⁶, inwhich R⁶ represents hydrogen or an aryl group substituent; for theaffinity binding of a prion protein.

In some embodiments, the ligand is a compound of formula (II),

wherein R1 represents a group —(CH₂)_(m)-Q¹, wherein m is from 0 to 7,and Q¹ represents —CR¹¹R¹²R¹³ or —NR¹¹R¹², in which R¹¹, R¹² and R¹³independently represent hydrogen, alkyl, cycloalkyl or heterocycloalkyl,or two of R¹¹, R¹² and R¹³, together with the carbon or nitrogen atom towhich they are attached, form an optionally substituted cycloalkyl oroptionally substituted heterocycloalkyl group; R² represents a group—(CH₂)_(n)-Q², wherein n is from 0 to 7, and Q² represents —CR²¹R²²R²³or —NR²¹R²², in which R²¹, R²² and R²³ independently represent hydrogen,alkyl, cycloalkyl or heterocycloalkyl, or two of R¹¹, R¹² and R¹³,together with the carbon or nitrogen atom to which they are attached,form an optionally substituted cycloalkyl or optionally substitutedheterocycloalkyl group, and R³ is hydrogen or an aryl group substituentor R³ is a solid support optionally attached via a spacer; Z representsan oxygen atom, a sulphur atom or NR⁴; Y represents an oxygen atom, asulphur atom or NR⁵; in which R⁴ and R⁵, which may be the same ordifferent, represent hydrogen, optionally substituted alkyl containing 1to 6 carbon atoms, optionally substituted phenyl, optionally substitutedbenzyl or optionally substituted β-phenylethyl; and one of X¹ and X²represents a nitrogen atom and the other of X¹ and X² represents anitrogen atom or CR⁶, in which R⁶ represents hydrogen or an aryl groupsubstituent; for the affinity binding of a prion protein.

In some embodiments, a) X¹ and X² are both nitrogen; b) both Z and Yrepresent NR⁴, in particular where R⁴ is hydrogen; c) m is from 0 to 4,more preferably from 0 to 3, and most preferably from 1 to 3; d) Q¹ is—NR¹¹R¹²; and R^(H) and R¹² preferably form, together with the nitrogenatom to which they are attached, a heterocycloalkyl group; e) n is from0 to 4, more preferably from 0 to 2, and most preferably is 0; and f)R²¹ and R²² form, together with the carbon atom or nitrogen atom towhich they are attached, a cycloalkyl or heterocycloalkyl group.

In some embodiments, Q² represents a hydrophobic cycloalkyl orheterocycloalkyl group, especially with ring systems that comprise atleast six atoms.

In some embodiments, Q¹ represents a heterocycloalkyl group, especiallypiperidyl, particularly 1-piperidyl, or piperazinyl, particularly1-piperazinyl.

In some embodiments, the ligand is a compound of formula (III),

wherein R1 represents an alkylene chain —(CH₂)_(p)—CH₃, wherein p isfrom 0 to 6, substituted by one or more carboxyl groups and optionallysubstituted by one or more further alkyl group substituents; R²represents a group —(CH₂)_(q)—Ar, wherein q is from 0 to 7, and Arrepresents an optionally substituted aryl group; and R³ is hydrogen oran aryl group substituent or R³ is a solid support optionally attachedvia a spacer; Z represents an oxygen atom, a sulphur atom or NR⁴; Yrepresents an oxygen atom, a sulphur atom or NR⁵; in which R⁴ and R⁵,which may be the same or different, represent hydrogen, optionallysubstituted alkyl containing 1 to 6 carbon atoms, optionally substitutedphenyl, optionally substituted benzyl or optionally substitutedβ-phenylethyl; and one of X¹ and X² represents a nitrogen atom and theother of X¹ and X² represents a nitrogen atom or CR⁶, in which R⁶represents hydrogen or an aryl group substituent; for the affinitybinding of a prion protein.

In some embodiments, a) X¹ and X² are both nitrogen; b) both Z and Yrepresent NR⁴, in particular where R⁴ is hydrogen; c) p is from 0 to 4,more preferably from 0 to 3; d) R¹ is substituted with one or twocarboxyl groups, at least one of those carboxyl groups being carried bythe terminal carbon atom of the alkylene chain —(CH₂)_(p)—CH₃; e) q isfrom 0 to 4, more preferably from 0 to 3, and most preferably is 1 or 2;and f) Ar is a monocyclic carbocyclic or heterocyclic aromatic group,optionally substituted by one or more substituents, selected from thegroup consisting of phenyl, phenoxy, tolyl, chlorobenzyl, methoxybenzyl,fluorobenzyl, pyridyl and indoyl.

In some embodiments, R¹ is carboxymethyl, 4-carboxybutyl or1-(1,3-dicarboxy)propyl.

In some embodiments, Ar is phenyl, 4-hydroxyphenyl or pyridyl,particularly 2-pyridyl.

In some embodiments, R³ is hydrogen or an aryl group substituent or R³is a solid support optionally attached via a spacer; X¹ and X² are bothN; Y and Z both represent NH; m represents 2; Q¹ represents piperidyl orpiperazinyl; n represents 0 or 2; and Q² represents 1-piperidyl oradamantyl.

In some embodiments, R³ is hydrogen or an aryl group substituent or R³is a solid support optionally attached via a spacer; X¹ and X² are bothN; Y and Z both represent NH; R¹ represents carboxymethyl,4-carboxybutyl and 1-(1,3-dicarboxy)propyl; q represents 2; and Arrepresents phenyl, 2-pyridyl or 4-hydroxyphenyl.

In some embodiments, the ligand is an inorganic compound or component,such as, but not limited to, aluminum (such as aluminum oxide) or silica(such as fused silica). In some embodiments, the inorganic compound isAl203 or SiO2.

In some embodiments, the ligand comprises one or more functional groups.The term “functional group” is used herein to denote chemical groups,subgroups, or substructures that impart characteristic chemical,physical, or physicochemical behaviors to a molecule or a material.Functional groups described herein include, but are not limited to,hydrophilic, such as positively, negatively or uncharged or neutral, orhydrophobic. Amphiphilic or multifunctional functional groups are alsoenvisioned and fall within the scope of the present invention.Functional groups include organic and inorganic functional groups.Preferred functional groups contain amine, phenyl or sulfite groups. Apreferred amine group is a primary, secondary, tertiary, or quaternaryammonium ion such as dimethylaminoethyl (DMAE) or trimethylaminoethyl(TMAE).

Other exemplary functional groups include, but are not limited to:—CH2—CHOH—CH2NH2; —C6H5; —(CH2)3-CH3; —CH2-CH2-NH(C2H5)2;—SO2-CH2-CH3′-CH2-CH2-H(CH3)2; —CH2-CH2-(CH3)3; —SO32-. Additionallyuseful functional groups include sulfonyl groups and tresyl groups. Itis to be understood that functional groups can be inherently present ina ligand, or can be added to the ligand by chemical modification.

In some embodiments, the ligand contains an amino group. These include,for example, amino resin, such as Toyopearl™ Amino-650M, Toyopearl™Amino-AMN31, TSK-GEL™-Amino 750C, or functional equivalents thereof. Insome embodiments, the ligand comprises a phenyl group. These include,for example, TSK-GEL™ Phenyl-5PW or functional equivalent thereof.

In some embodiments, the ligand is a polymeric material, such aschromatographic resins (for example amino resins), that bind withselectivity and specificity to prion proteins. An example ofchromatographic resin is PRDT Prion Reduction Resins (ProMeticBiosciences, Ltd, 211 Cambridge Science Park, Milton Road, Cambridge,CB4 0WA, UK). In some embodiments, the polymeric material contains oneor more functional groups, such as functional groups described above. Insome embodiments, the ligand is a polymeric material having amethacrylate backbone, such as, but not limited to, a commerciallyavailable TSK, TOYOPEARL, or FACTOGEL resin (Tosoh Bioscience,Montgomeryville, Pa.).

Other ligands that can be used in methods of the present inventioninclude, for example, ligands that interact with amyloid plaque e.g.,Congo:E;Led (Ingrosso, L., et al., Congo Red: Prolongs the IncubationPeriod in Scrapie-infected Hamsters. Virology 69:506-508 (1995));1,4-iodo, 4-deoxy doxorubicin (Tagliavini, F., et al., Effectiveness ofAnthracycline Against Experimental Prion Diseases in Syrian Hamsters.Science 276:1119-1122 (1997)); amphotericin 13, porphyrins andphthalocyanines (Priola, S. A., et al., Porphyrin and PhthalocyanineAntiscrapie Compounds, Science 287:1503-1506 (2000)); metals (Stocker etal., Biochemistry, 37, 7185-7193 (1998)); peptides that interact withPrP to form complexes (see U.S. Pat. No. 5,750,361 to Prusiner et al.and Solo, C. et al., Reversion of Prion Protein Conformational Changesin Synthetic p-sheet Breaker Peptides, Lances, 355:192-197 (2000));heparin and other polysulphated polyanions (Caughey, B., et al., Bindingof the Protease-sensitive Form of Prion Protein PrP to SulphatedGlycosaminoglycan and Congo Red, J. Virology 68:2135-2141(1994));antibodies (Kascsak, R. J., et al., Immunodiagnosis of Prion Disease,Immunological Invest. 26:259-268 (1997)); and other proteins, e.g.plasminogen (Fischer, M. B. et al., Binding of Disease-associated PrionProtein to: Plasminogen., Nature 408:479-483 (2000)).

The ligands may be attached to any supporting materials. “Supportmaterial” used herein refer to any compound or material which mayprovide a physical or chemical means of separating the ligand andproteins bound thereto from the rest of the composition. The supportingmaterial may be particulate or non-particulate, soluble or insoluble,porous or non-porous.

Examples of support materials include, but not limited to, naturallyoccurring polymers, e.g., a polysaccharide such as agarose, alginate,carrageenan, chitin, cellulose, dextran or starch; synthetic polymerssuch as polyacrylamide, polystyrene, polyacrolein, polyvinyl alcohol,polymethylacrylate, perfluorocarbon; inorganic compounds such as silica,glass, kieselquhr, alumina, iron oxide or other metal oxides, orcopolymers consisting of any combination of two or more naturallyoccurring polymers, synthetic polymers or inorganic compounds. Alsocontemplated are soluble support materials comprising polymers such asdextran, polyethylene glycol, polyvinyl alcohol or hydrolysed starchwhich provide affinity-ligand matrix conjugates for use in liquidpartitioning

In some embodiments, the supporting material is a solid support such asa column, a bead, a membrane, a cartridge, a filter, a dipstick, amicrotiter plate, a test tube, solid powder, a cast or extrusion moldedmodule, a mesh, a magnetic particle composite, or any other solidmaterials. The solid materials may be coated with a substance such aspolyethylene, polypropylene, poly(4-methylbutene), polystyrene,polyacrylate, polyethylene terephthalate, rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF), silicones,polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and thelike. Alternatively, substances that form gels, such as proteins (e.g.gelatins), lipopolysaccharides, silicates, agarose and polyacrylamidesare used. Polymers such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like can also be used. The ligands areattached to or dispersed throughout the support materials.

In some embodiments, the supporting material is activated agarose,silica, cellulose, glass, methacrylate, hydroxyethylmethacrylate,polyacrylamide, styrenedivinylbenzene, Hyper D or perfluorocarbons. Insome embodiments, the supporting material is a methacrylate material, ofthe type sold under the trade name Toyopearl (available from TosohBioscience LLC, 156 Keystone Drive, Montgomeryville, Pa. I 18936, USA).WO 97/10887 describes methods of attaching affinity ligands to supportmatrices, e.g. the use of activating methods, and methods of attachingthe affinity ligand to a matrix via a spacer, e.g. by condensationreactions, to form affinity ligand-matrix conjugates.

In some embodiments, the ligands and/or supporting materials arereusable. In some embodiments, the ligands and/or supporting materialsfor single use only.

The prion binding capacity of a ligand can be evaluated by determiningthe infectious titre in a ligand. For example, a dilution series of areference stock is prepared and tested in the standard Western blotassay. The titres observed from the reference stock are compared withthe corresponding titres observed in a bioassay. The Western blot titrescan then be converted into infectious titres using the formula:Titre_([Bioassay])=Titre_([WesternBlot])+(intercept of a linearregression analysis)/(slope of a linear regression analysis). Once theinfectious titre per ml is calculated, the total prion protein bound tothe column can be determined. The capacity of the prion binding of aligand is determined using the amount of prion protein observed directlybound to the ligand. In some embodiments, the prion binding capacity ofa ligand is at least about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.5, or 10.0 log₁₀ ID₅₀ per mlof ligand. In some embodiments, the prion binding capacity of a ligandis at least about 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ ID₅₀ per ml ofligand.

Method of Making Nanoparticles

Methods of making compositions containing albumins and substantiallywater insoluble pharmacologically active agents are known in the art.For example, nanoparticles containing substantially water insolublepharmacologically active agents and albumins can be prepared underconditions of high shear forces (e.g., sonication, high pressurehomogenization, or the like). These methods are disclosed in, forexample, U.S. Pat. Nos. 5,916,596; 6,506,405; 6,749,868, and 6,537,579and also in U.S. Pat. Pub. Nos. 2005/0004002 and 2007/0082838, and PCTPublication W099/00113, which are each hereby incorporated by referencein their entireties.

In one exemplary embodiment, the substantially water insolublepharmacologically active agent (e.g., paclitaxel) is dissolved in anorganic solvent. Suitable organic solvents include, for example,ketones, esters, ethers, chlorinated solvents, and other solvents knownin the art. For example, the organic solvent can be methylene chloride.In some embodiments, the organic solvent can be a mixture of a waterimmiscible solvent (such as chloroform) and a water miscible solvent(such as a water miscible alcohol solvent, such as chloroform/methanol,chloroform/ethanol, chloroform/propanol, or chloroform/t-butanol (forexample with a ratio (v/v) of about any of 1:9, 1:8, 1:7, 1:6, 1:5, 1:4,1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1 or with a ratio(v/v) of about any of 3:7, 5:7, 4:6, 5:5, 6:5, 8:5, 9:5, 9.5:5, 5:3,7:3, 6:4, or 9.5:0.5). The solution is added to an albumin (e.g., humanserum albumin) The mixture is subjected to high pressure homogenization(e.g., using standard homogenization devices). The emulsion may becycled through the high pressure homogenizer for between about 2 toabout 100 cycles, such as about 5 to about 50 cycles or about 8 to about20 cycles (e.g., about any of 8, 10, 12, 14, 16, 18 or 20 cycles). Theorganic solvent can then be removed by evaporation utilizing suitableequipment known for this purpose, including, but not limited to, rotaryevaporators, thin file evaporators, falling film evaporators, wiped filmevaporators, spray driers, and the like. The solvent may be removed, forexample, at reduced pressure (such as at about any of 5 mm Hg, 10 mm Hg,15 mm Hg, 20 mm Hg, 25 mm Hg, 30 mm Hg, 40 mm Hg, 50 mm Hg, 100 mm Hg,200 mm Hg, or 300 mm Hg). The amount of time used to remove the solventunder reduced pressure may be adjusted based on the volume of theformulation. For example, for a formulation produced on a 300 mL scale,the solvent can be removed at about 1 to about 300 mm Hg (e.g., aboutany of 5-100 mm Hg, 10-50 mm Hg, 20-40 mm Hg, or 25 mm Hg) for about 1to about 120 minutes, including about 5 to about 60 minutes (e.g., aboutany of 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 18, 20, 25, or 30 minutes).

If desired, albumin solution (such as prion-removed albumin solution)may be added to the dispersion to adjust the albumin to drug (e.g.,paclitaxel) ratio or to adjust the concentration of the taxane (e.g.,paclitaxel) in the dispersion. For example, albumin solution (e.g., 25%w/v) can be added to adjust the albumin to substantially water insolublepharmacologically active agent (e.g., paclitaxel) ratio to about any of18:1, 15,:1 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7.5:1, 7:1, 6:1,5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18. For example, albuminsolution (e.g., 25% w/v) or another solution is added to adjust theconcentration of the substantially water insoluble pharmacologicallyactive agent (e.g., paclitaxel) in the dispersion to about any of 0.5mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25mg/ml, 30 mg/ml, 40 mg/ml, or 50 mg/ml. The dispersion may beindividually or serially filtered through one or more filters, such as1.2 μm, 0.8 μm, 0.45 μm, and 0.22 μm filters; combinations of two ormore thereof, or the combination with any other filters known in theart.

If desired, a second therapy (e.g., one or more compounds useful fortreating cancer), an antimicrobial agent (such as citrate or edetate),sugar (such as sucrose), and/or stabilizing agent can also be includedin the composition. This additional agent can either be admixed with thesubstantially water insoluble pharmacologically active agent (e.g.,paclitaxel) and/or the albumin during preparation of the composition, oradded after the nanoparticle composition is prepared. In someembodiments, the agent is admixed with the nanoparticle compositionprior to lyophilization. In some embodiments, the agent is added to thelyophilized composition. In some embodiments when the addition of theagent changes the pH of the composition, the pH in the composition aregenerally (but not necessarily) adjusted to a desired pH. Exemplary pHvalues of the compositions include, for example, in the range of about 5to about 8.5. In some embodiments, the pH of the composition is adjustedto no less than about 6, including for example no less than any of about6.5, 7, or 8 (e.g., about 8).

As discussed below in more detail, the prion-removal process can becarried out concurrently with the manufacturing process. For example,the prion-removal process can be carried out after the mixture ofalbumin and substantially water insoluble pharmacologically active agentis formed and prior to subjecting the mixture to the high shearcondition. In some embodiments, the prion-removal process can be carriedout after the mixture has been subjected to the high shear condition andprior to the removal of the organic solvent. In some embodiments, theprion-removal process is carried out after the removal of the organicsolvent. In some embodiments, additional albumin is added to thepost-evaporation suspension prior to the prion-removal process. In someembodiments, the prion-removal process is carried out under a sterilecondition. In some embodiments, the prion-removal process is carried outby using a cartridge which simultaneously sterile filters thecomposition.

Method of Removing Prions from Nanoparticle Compositions or IntermediateCompositions

The present invention in one aspect provides methods comprising removingprion proteins from a nanoparticle composition, such as nanoparticlecompositions formed by methods described above. In another aspect thereare provided methods of removing prion proteins from intermediatecompositions generated during the nanoparticle manufacture process(hereinafter referred to as “the intermediate composition”).

Generally, the methods comprise contacting a composition comprisingalbumin and a substantially water insoluble pharmacologically activeagent with a ligand capable of binding to a prion protein, and removingthe ligand and protein bound thereto from the nanoparticle composition.This process can be repeated one or more times, with the same or adifferent ligand. Two or more ligands can also be used simultaneouslyduring the prion removal process.

In some embodiments, the nanoparticle composition or the intermediatecomposition has a prion infectivity of about 100 IU-ic/ml, 90IU-ic/ml,50IU-ic/ml, or 10IU-ic/ml.

During the prion-removal process, the ligand is brought into contactwith the nanoparticle composition or intermediate composition andallowed to bind to prion proteins in the nanoparticle composition or theintermediate composition. Conditions suitable for the binding can bedetermined and optimized to facilitate binding of the ligand to a prionprotein based on the nature of the ligand and its binding specificity tothe prion protein. In some embodiments, the binding is carried out at atemperature of about 0° C. to about 39° C., including for example about20° C. to about 25° C. The binding can be carried out at pH of about 4to about 10, including for example about 5 to about 9, about 6 to about8, about 6.8 to about 7.5, about 6.9 to about 7.4,or about 7.Optionally, blocking agents can be used to reduce non-specific bindingto the ligand.

After the contacting step, the ligand and proteins bound thereto areremoved from the rest of the composition. The removal can be carried outin a variety of ways, depending on the nature of the ligand and thesupporting material (if any) used to facilitate the separation. Forexample, the ligand and proteins bound thereto can be separated out bychromatography, such as, but not limited to, thin-layer, column andbatch chromatography; solid support and membrane separation; reactorseparation; magnetic separation, immunoseparation; and colloidalseparation.

In some embodiments, ligand may be immobilized on a support such as abead or a membrane, which in turn is allowed to contact the nanoparticlecomposition or the intermediate composition. Ligand-immobilized supportis allowed to contact the nanoparticle composition or the intermediatecomposition under a condition sufficient to cause formation of aprion-ligand complex. The solid phase is then separated from thecomposition, thereby removing the prion protein bound to the ligand fromthe sample. For example, in one exemplary embodiment, the ligands areimmobilized to a column, such as a chromatography column, a sample (suchas the nanoparticle composition) is then passed through the columneither due to the force of gravity or under pressure, such as in a highpressure liquid chromatography column. Prion proteins in the sample willbind to the ligand immobilized on the column, and the sample passingthrough can be collected. This process can repeat several times toachieve the desired result.

The flow rate of a sample (such as nanoparticle composition orintermediate composition) in a column can be adjusted to maximize thebinding of the ligand and the prion proteins in a sample. In someembodiments, the binding is carried out at a flow rate of about 0.1 mlper minute to about 5.0 ml per minute, about 0.1 ml per minute to about2.5 ml per minute, about 0.1 ml per minute to about 0.25 ml per minute,about 0.25 per minute to about 0.5 ml per minute, about 0.5 ml perminute to about 1.0 ml per minute, about 1.0 ml per minute to about 1.5ml per minute, about 1.5 ml per minute to about 2.0 ml per minute, about2.0 ml per minute to about 2.5 ml per minute, about 2.5 ml per minute toabout 3.0 ml per minute, about 3.0 ml per minute to about 3.5 ml perminute, about 3.5 ml per minute to about 4.0 ml per minute, about 4.0 mlper minute to about 4.5 ml per minute, or about 4.5 ml per minute toabout 5.0 ml per minute, including for example about 0.1 ml per minute,0.25 ml per minute, 0.5 ml per minute, 1.0 ml per minute, 1.5 ml perminute, 1.7 ml per minute, 1.8 ml per minute, 1.9 ml per minute, 2.0 mlper minute, 2.1 ml per minute, 2.3 ml per minute, 2.5 ml per minute, 2.7ml per minute, 3.0 ml per minute, 3.5 ml per minute, 4.0 ml per minute,4.5 ml per minute, or 5.0 ml per minute. In some embodiments, the flowrate is at least about 10 ml per minute, such as at least about any of20 ml per minute, 30 ml per minute, 40 ml per minute, 50 ml per minute.

The total flow-through volume or total flow-through time during abinding process can also be adjusted to maximize the binding of theligand and the prion proteins in a sample (such as nanoparticlecomposition or the intermediate composition). In some embodiments, thetotal flow-through volume is about 50 times to about 1000 times of thecolumn volume, including for example about 50 times to about 500 timesof the column volume, about 100 times to about 600 times of the columnvolume, or about 200 times to about 800 times of the column volume. Insome embodiments, the total flow-through volume is about 100 times ofthe column volume. In other embodiments, the total flow-through volumeis about 500 times of the column volume. In some embodiments, the totalflow-through time is about 1 hour to about 30 hours, including forexample about 2 hours to about 25 hours, about 3 hours to about 20hours, or about 3 hours to about 17 hours. In some embodiments, thetotal flow-through time is any of about 3 hours, 4 hours, 6 hours, 8hours, 10 hours, 12 hours, 14 hours, 16 hours, 17 hours, 18 hours, 19hours, or 20 hours. In some embodiments, the total flow through time ismore than about 24 hours. In some embodiments, the total flow throughtime is less than about 8, including for example any of 7, 6, 5, 4, 3,2, 1, or 0.5 hours.

Alternatively, the ligand can first be brought into contact with thenanoparticle composition or the intermediate composition, under acondition sufficient to cause formation of a prion-ligand complex. Theprion-ligand complex is then subsequently removed by using a column,such as an affinity-based chromatography. To facilitate the separation,the ligand may be conjugated to a binding partner so that theligand/prion complex can be removed by using an affinity columncontaining a molecule that recognizes the binding partner.

In addition to batch or column chromatography, a variety ofconfigurations, modifications and variations of the use of the ligandsfor binding prion proteins are also envisioned. Such variations andmodifications include, but are not limited to: batch processes,continuous processes, moving bed chromatography processes; low, medium,or high pressure processes; or small, medium or large scale processes.In some embodiments, the ligands are on a membrane, fibers bead,impregnated into a non-woven mesh, or coating fibers contained within afilter housing.

In some embodiments, the removal step does not significantly result inyield loss and/or change in the property and/or stability of the albuminFor practical purposes, the recovery of the albumin in its originalbiological state should be substantially maintained at least to a levelin excess of 50%, including for example 80%, or 90%, or more. In someembodiments, the recovery rate of albumin from the prion process ishigher than any of about 80%, 90%, 95%, or 99%. In some embodiments, theconcentration of albumin in the nanoparticle composition is adjusted orcontrolled prior to the prion removal step in order to minimizenon-specific binding and loss of albumin during the process. Forexample, the concentration of albumin can be in the range of about 1% toabout 50%, about 5% to about 25%, about 5% to about 30%, about 5% toabout 40%, about 5% to about 10%, about 10% to about 15%, about 15% toabout 20%, about 20% to about 25%, about 25% to about 30% etc.,including for example about 5%, 10%, 15%, 20%, 25%, or 30% albumin.

In some embodiments, the removal step does not significantly result inyield loss and/or change in the property and/or stability of thenanoparticles in the composition.

In some embodiments, the removal step does not significantly result inyield loss and/or change in the property or loading of the substantiallywater insoluble pharmacologically active agent in the composition.

In some embodiments, the removal step does not significantly result inchange in the ratio of albumin to the substantially water insolublepharmacological agent in the composition.

The prion-removed composition can be analyzed to determine the clearancerate of the prion removal process. The ligand with bound prion proteinsmay also be analyzed (directly or after elution) to determine theclearance rate.

The removal of prion protein can be determined based on reduction in theprion protein level and/or a reduction in infectivity. In someembodiments, at least about 50%, including for example at least about60%, 70%, 80%, 90%, 95%, 99%, or 100% of the prion proteins are removedfrom the nanoparticle composition. In some embodiments, the infectivityof the post-removal albumin composition is at least about 10×, 20×, 30×,40×, 50×, 80×, 100×, 200×, 500×, 1000×, 10⁴×, 10⁵×, 10⁶× less than thatof the nanoparticle composition.

In some embodiments, serial infectivity is used to determine theclearance rate of the prion removal process. Serial dilutions of asamples are made and dilutions are examined for infectious activity, forexample in an assay animal The dilution at which half of the animalsbecome infected is the infectious titer. For example, if a 5 folddilution is required, the sample may be defined as having 5 logs ofinfectivity. By comparing the log infectivity of the nanoparticlecomposition and that of the post-removal nanoparticle composition, onecan determine the clearance rate of the prion removal process. In someembodiments, the prion removal method results in a reduction of any oneof 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, or 4, 5, 6, 7,8, 9, or 10 logs of infectivity.

In some embodiments, the clearance rate of the prion-removal process isdetermined based on spiking experiments with infectious materials byfollowing steps described herein for the prion-removal method. Suitablespiking agents include, but are not limited to, brain homogenates,microsomes, caveolae-like domains, purified PrPsc, and prion fibrils. Insome embodiments, the spiking agent is detergent solubilized (such assarkosyl solubilized). In some embodiments, the spiking ratio in thecomposition is in the range of about 0.001% to about 5%, about 0.001% toabout 0.25%, about 0.001% to about 0.1%, about 0.001% to about 0.005%,about 0.005% to about 0.075%, about 0.075% to about 0.01%, about 0.01%to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 0.75%,about 0.75% to about 1%, about 1% to about 2%, about 2% to about 3%, orabout 3% to about 5%, including for example about 0.001%, 0.005%,0.075%, 0.01%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, or 5%.

In some embodiments, the reduction factors (RF) is used to determine theclearance rate of the prion removal process. The RF can be calculatedusing the formula:

RF=(V ₁ ×T ₁)/(V ₂ ×T ₂)

or

Log₁₀[RF]=[ Log₁₀(V ₁)+Log₁₀(T ₁)]−[ Log₁₀(V ₂)+Log₁₀(T ₂)].

Wherein V₁ and T₁ are the volume and titre of the initial albumincomposition, respectively, and V₂ and T₂ are the volume and titre of thepost-removal albumin composition. Reduction factors can be rounded to 1decimal place after the final calculation. In some embodiments, areduction factor of at least about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 log₁₀ infectivity of the prionproteins is removed from the initial albumin composition. For example,the prion protein can be removed by a reduction factor of greater thanor equal to 2.5 log₁₀ in a 0.5% sarkosyl solubilized fraction spikedinto a 20% albumin composition. As another example, the prion proteincan also be removed by a reduction factor of greater than or equal to2.0 log₁₀ in a 0.5% sarkosyl solubilized fraction spiked into a 25%albumin composition.

The removal of prions can be evaluated by standard Western blotanalysis. For example, the post-binding ligands can first be treatedwith Proteinase K, which digests all PrPc but not PrPsc. The digest isthen run on SDS gel and transblotted to a sheet of nitrocellulose orPVDF membrane. The separated PrPsc bands are then visualized using 3F4or 6H4. 3F4 reacts with residues 109-112 PrP from humans, hamsters, andfelines. In one exemplary embodiment, incubation was carried out at aconcentration of 0.6 ug/ml for a minimum of one hour, after which excessantibody was washed away and the membranes incubated with a rabbitanti-mouse horse-radish peroxidase conjugate (1:1000 dilution) for aminimum of one hour. After extensive washing with TTBS, the membraneswere developed using enhanced chemiluminescence. In some embodiments,the removal of prion proteins is evaluated according to the Guidelinefor the Investigation of Manufacturing Processes for Plasma-DerivedMedicinal Products with Regard to vCJD Risk (CPMP5136/03).

Also contemplated herein are compositions made during the prion removalprocess. Thus, in some embodiments, there is provided a compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, further comprising a ligandcapable of binding to a prion protein. In some embodiments, there isprovided a mixture comprising nanoparticle comprising albumin and asubstantially water insoluble pharmacologically active agent, and aligand capable of binding to a prion protein attached to a supportmaterial, such as one or more supporting materials described herein. Insome embodiments, there is provided a column loaded with a compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, wherein the column comprises aligand capable of binding to a prion protein. In some embodiments, thereis provided a composition comprising nanoparticles comprising albuminand a substantially water insoluble pharmacologically active agent and aresin comprising a ligand capable of binding to a prior protein. Forexample, in some embodiments, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent and a DVR resin. In some embodiments,there is provided a composition comprising nanoparticles comprisingalbumin and a substantially water insoluble pharmacologically activeagent and a PRDT resin.

In some embodiments, there is provided a composition comprising amixture of an aqueous albumin solution and a substantially waterinsoluble pharmacologically active agent dispersed in an organicsolvent, further comprising a ligand capable of binding to a prionprotein. In some embodiments, there is provided a composition comprisinga mixture of an aqueous albumin solution and a substantially waterinsoluble pharmacologically active agent dispersed in an organicsolvent, and a ligand capable of binding to a prion protein attached toa support material, such as one or more supporting materials describedherein. In some embodiments, there is provided a column loaded with acomposition comprising a mixture of an aqueous albumin solution and asubstantially water insoluble pharmacologically active agent dispersedin an organic solvent, wherein the column comprises a ligand capable ofbinding to a prion protein.

Also provided are compositions made by methods described herein. Thecomposition in some embodiments is bioequivalent to a composition notsubject to prion-removal process.

Nanoparticle Compositions

The nanoparticle compositions described herein comprise nanoparticlescomprising (in various embodiments consisting essentially of) albuminand a substantially water insoluble pharmacologically active agent (suchas paclitaxel). Nanoparticles of poorly water soluble drugs (such astaxane) have been disclosed in, for example, U.S. Pat. Nos. 5,916,596;6,506,405; 6,749,868, and 6,537,579 and also in U.S. Pat. Pub. Nos.2005/0004002 and 2007/0082838, and PCT Patent Application WO08/137148,the content of each of which is incorporated herein in their entirety.

In some embodiments, the composition comprises nanoparticles with anaverage or mean diameter of no greater than about 1000 nanometers (nm),such as no greater than about any of 900, 800, 700, 600, 500, 400, 300,200, and 100 nm. In some embodiments, the average or mean diameters ofthe nanoparticles is no greater than about 200 nm. In some embodiments,the average or mean diameters of the nanoparticles is no greater thanabout 150 nm. In some embodiments, the average or mean diameters of thenanoparticles is no greater than about 100 nm. In some embodiments, theaverage or mean diameter of the nanoparticles is about 20 to about 400nm. In some embodiments, the average or mean diameter of thenanoparticles is about 40 to about 200 nm In some embodiments, thenanoparticles are sterile-filterable.

In some embodiments, the nanoparticles in the composition describedherein have an average diameter of no greater than about 200 nm,including for example no greater than about any one of 190, 180, 170,160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 nm. In someembodiments, at least about 50% (for example at least about any one of60%, 70%, 80%, 90%, 95%, or 99%) of all the nanoparticles in thecomposition have a diameter of no greater than about 200 nm, includingfor example no greater than about any one of 190, 180, 170, 160, 150,140, 130, 120, 110, 100, 90, 80, 70, or 60 nm. In some embodiments, atleast about 50% (for example at least any one of 60%, 70%, 80%, 90%,95%, or 99%) of all the nanoparticles in the composition fall within therange of about 20 to about 200 nm, including for example any one ofabout 30 to about 180 nm, and any one of about 40 to about 150, about 50to about 120, and about 60 to about 100 nm

In some embodiments, at least about 5% (including for example at leastabout any one of 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or90%) of the album in the nanoparticle portion of the composition arecrosslinked (for example crosslinked through one or more disulfidebonds).

In some embodiments, the nanoparticles comprise the substantially waterinsoluble pharmacologically active agent (such as paclitaxel) coatedwith an albumin (e.g., human serum albumin). In some embodiments, thecomposition comprises substantially water insoluble pharmacologicallyactive agent in non-nanoparticle form, wherein at least about any one of50%, 60%, 70%, 80%, 90%, 95%, or 99% of the substantially waterinsoluble pharmacologically active agent in the composition are innanoparticle form. In some embodiments, the substantially waterinsoluble pharmacologically active agent in the nanoparticlesconstitutes more than about any one of 50%, 60%, 70%, 80%, 90%, 95%, or99% of the nanoparticles by weight. In some embodiments, thenanoparticles have a non-polymeric matrix. In some embodiments, thenanoparticles comprise a core of substantially water insolublepharmacologically active agent that is substantially free of polymericmaterials (such as polymeric matrix).

In some embodiments, the composition comprises albumin in bothnanoparticle and non-nanoparticle portions of the composition, whereinat least about any one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of thealbumin in the composition are in the non-nanoparticle portion of thecomposition.

In some embodiments, the nanoparticle composition is substantially free(such as free) of surfactants (such as Cremophor®, Tween 80, or otherorganic solvents used for the administration of substantially waterinsoluble pharmacologically active agents). In some embodiments, thenanoparticle composition contains less than about any one of 20%, 15%,10%, 7.5%, 5%, 2.5%, 1% or less organic solvent. In some embodiments,the weight ratio of albumin and substantially water insolublepharmacologically active agent in the nanoparticle composition is about18:1 or less, such as about 15:1 or less, for example about 10:1 orless. In some embodiments, the weight ratio of albumin and substantiallywater insoluble pharmacologically active agent in the composition fallswithin the range of any one of about 1:1 to about 18:1, about 2:1 toabout 15:1, about 3:1 to about 13:1, about 4:1 to about 12:1, about 5:1to about 10:1. In some embodiments, the weight ratio of albumin andsubstantially water insoluble pharmacologically active agent in thenanoparticle portion of the composition is about any one of 1:2, 1:3,1:4, 1:5, 1:10, 1:15, or less. In some embodiments, the weight ratio ofthe albumin (such as human serum albumin) and the substantially waterinsoluble pharmacologically active agent in the composition is any oneof the following: about 1:1 to about 18:1, about 1:1 to about 15:1,about 1:1 to about 12:1, about 1:1 to about 10:1, about 1:1 to about9:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about6:1, about 1:1 to about 5:1, about 1:1 to about 4.1, about 1:1 to about3:1, about 1:1 to about 2:1, or about 1:1.

In some embodiments, the nanoparticle composition comprises one or moreof the above characteristics.

The nanoparticles described herein may be present in a dry formulation(such as lyophilized composition) or suspended in a biocompatiblemedium. Suitable biocompatible media include, but are not limited to,water, buffered aqueous media, saline, buffered saline, optionallybuffered solutions of amino acids, optionally buffered solutions ofproteins, optionally buffered solutions of sugars, optionally bufferedsolutions of vitamins, optionally buffered solutions of syntheticpolymers, lipid-containing emulsions, and the like.

In some embodiments, the pharmaceutically acceptable carrier compriseshuman serum albumin Human serum albumin (HSA) is a highly solubleglobular protein of Mr 65K and consists of 585 amino acids. HSA is themost abundant protein in the plasma and accounts for 70-80% of thecolloid osmotic pressure of human plasma. The amino acid sequence of HSAcontains a total of 17 disulphide bridges, one free thiol (Cys 34), anda single tryptophan (Trp 214). Intravenous use of HSA solution has beenindicated for the prevention and treatment of hypovolumic shock and inconjunction with exchange transfusion in the treatment of neonatalhyperbilirubinemia. Other albumins are contemplated, such as bovineserum albumin Use of such non-human albumins could be appropriate, forexample, in the context of use of these compositions in non-humanmammals, such as the veterinary (including domestic pets andagricultural context).

Human serum albumin (HSA) has multiple hydrophobic binding sites (atotal of eight for fatty acids, an endogenous ligand of HSA) and binds adiverse set of substantially water insoluble pharmacologically activeagents, especially neutral and negatively charged hydrophobic compounds.Two high affinity binding sites have been proposed in subdomains IIA andIIIA of HSA, which are highly elongated hydrophobic pockets with chargedlysine and arginine residues near the surface which function asattachment points for polar ligand features.

The albumin in the composition generally serves as a carrier for thesubstantially water insoluble pharmacologically active agent, i.e., thealbumin in the composition makes the substantially water insolublepharmacologically active agent more readily suspendable in an aqueousmedium or helps maintain the suspension as compared to compositions notcomprising an albumin This can avoid the use of toxic solvents (orsurfactants) for solubilizing the substantially water insolublepharmacologically active agent, and thereby can reduce one or more sideeffects of administration of the substantially water insolublepharmacologically active agent into an individual (such as a human).Thus, in some embodiments, the composition described herein issubstantially free (such as free) of surfactants, such as Cremophor(including Cremophor EL® (BASF)). In some embodiments, the nanoparticlecomposition is substantially free (such as free) of surfactants. Acomposition is “substantially free of Cremophor” or “substantially freeof surfactant” if the amount of Cremophor or surfactant in thecomposition is not sufficient to cause one or more side effect(s) in anindividual when the nanoparticle composition is administered to theindividual.

The amount of albumin in the composition described herein will varydepending on other components in the composition. In some embodiments,the composition comprises an albumin in an amount that is sufficient tostabilize the substantially water insoluble pharmacologically activeagent in an aqueous suspension, for example, in the form of a stablecolloidal suspension (such as a stable suspension of nanoparticles). Insome embodiments, the albumin is in an amount that reduces thesedimentation rate of the substantially water insolublepharmacologically active agent in an aqueous medium. Forparticle-containing compositions, the amount of the albumin also dependson the size and density of nanoparticles of the substantially waterinsoluble pharmacologically active agent.

A substantially water insoluble pharmacologically active agent is“stabilized” in an aqueous suspension if it remains suspended in anaqueous medium (such as without visible precipitation or sedimentation)for an extended period of time, such as for at least about any of 0.1,0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60,or 72 hours. The suspension is generally, but not necessarily, suitablefor administration to an individual (such as human). Stability of thesuspension is generally (but not necessarily) evaluated at a storagetemperature (such as room temperature (such as 20-25° C.) orrefrigerated conditions (such as 4° C.)). For example, a suspension isstable at a storage temperature if it exhibits no flocculation orparticle agglomeration visible to the naked eye or when viewed under theoptical microscope at 1000 times, at about fifteen minutes afterpreparation of the suspension. Stability can also be evaluated underaccelerated testing conditions, such as at a temperature that is higherthan about 40° C.

In some embodiments, the albumin is present in an amount that issufficient to stabilize the substantially water insolublepharmacologically active agent in an aqueous suspension at a certainconcentration. For example, the concentration of the substantially waterinsoluble pharmacologically active agent in the composition is about 0.1to about 100 mg/ml, including for example any of about 0.1 to about 50mg/ml, about 0.1 to about 20 mg/ml, about 1 to about 10 mg/ml, about 2mg/ml to about 8 mg/ml, about 4 to about 6 mg/ml, about 5 mg /ml. Insome embodiments, the concentration of the substantially water insolublepharmacologically active agent is at least about any of 1.3 mg/ml, 1.5mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, and50 mg/ml. In some embodiments, the albumin is present in an amount thatavoids use of surfactants (such as Cremophor), so that the compositionis free or substantially free of surfactant (such as Cremophor).

In some embodiments, the composition, in liquid form, comprises fromabout 0.1% to about 50% (w/v) (e.g. about 0.5% (w/v), about 5% (w/v),about 10% (w/v), about 15% (w/v), about 20% (w/v), about 25% (w/v),about 30% (w/v), about 40% (w/v), or about 50% (w/v)) of albumin In someembodiments, the composition, in liquid form, comprises about 0.5% toabout 5% (w/v) of albumin

In some embodiments, the weight ratio of albumin, e.g., albumin, to thesubstantially water insoluble pharmacologically active agent in thenanoparticle composition is such that a sufficient amount ofsubstantially water insoluble pharmacologically active agent binds to,or is transported by, the cell. While the weight ratio of albumin tosubstantially water insoluble pharmacologically active agent will haveto be optimized for different albumin and substantially water insolublepharmacologically active agent combinations, generally the weight ratioof albumin, e.g., albumin, to substantially water insolublepharmacologically active agent (w/w) is about 0.01:1 to about 100:1,about 0.02:1 to about 50:1, about 0.05:1 to about 20:1, about 0.1:1 toabout 20:1, about 1:1 to about 18:1, about 2:1 to about 15:1, about 3:1to about 12:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about9:1. In some embodiments, the albumin to substantially water insolublepharmacologically active agent weight ratio is about any of 18:1 orless, 15:1 or less, 14:1 or less, 13:1 or less, 12:1 or less, 11:1 orless, 10:1 or less, 9:1 or less, 8:1 or less, 7:1 or less, 6:1 or less,5:1 or less, 4:1 or less, and 3:1 or less.

In some embodiments, the albumin allows the composition to beadministered to an individual (such as human) without significant sideeffects. In some embodiments, the albumin is in an amount that iseffective to reduce one or more side effects of administration of thesubstantially water insoluble pharmacologically active agent to a human.The term “reducing one or more side effects of administration of thesubstantially water insoluble pharmacologically active agent” refers toreduction, alleviation, elimination, or avoidance of one or moreundesirable effects caused by the substantially water insolublepharmacologically active agent, as well as side effects caused bydelivery vehicles (such as solvents that render the substantially waterinsoluble pharmacologically active agents suitable for injection) usedto deliver the substantially water insoluble pharmacologically activeagent. Such side effects include, for example, myelosuppression,neurotoxicity, hypersensitivity, inflammation, venous irritation,phlebitis, pain, skin irritation, peripheral neuropathy, neutropenicfever, anaphylactic reaction, venous thrombosis, extravasation, andcombinations thereof. These side effects, however, are merely exemplaryand other side effects, or combination of side effects, associated withsubstantially water insoluble pharmacologically active agents can bereduced.

In some embodiments, the composition comprises Abraxane® (orNab-paclitaxel). In some embodiments, the composition is Abraxane® (orNab-paclitaxel). Abraxane° is a formulation of paclitaxel stabilized byhuman albumin USP, which can be dispersed in directly injectablephysiological solution. When dispersed in a suitable aqueous medium suchas 0.9% sodium chloride injection or 5% dextrose injection, Abraxane™forms a stable colloidal suspension of paclitaxel. The mean particlesize of the nanoparticles in the colloidal suspension is about 130nanometers. Since HSA is freely soluble in water, Abraxane™ can bereconstituted in a wide range of concentrations ranging from dilute (0.1mg/ml paclitaxel) to concentrated (20 mg/ml paclitaxel), including forexample about 2 mg/ml to about 8 mg/ml, about 5 mg/ml.

Substantially Water Insoluble Pharmacologically Active Agent

The compositions described herein comprise substantially water insolublepharmacologically active agents. For example, the solubility in water ofthe poorly water soluble agent at about 20-25° C. may be less than about10 mg/ml, including for example less than about any of 5, 2, 1, 0.5,0.2, 0.1, 0.05, 0.02, or 0.01 mg/ml. In some embodiments, thesubstantially water insoluble pharmacologically active agent is a solid.In some embodiments, the substantially water insoluble pharmacologicallyactive agent is a liquid. Substantially water insolublepharmacologically active agents described herein can be, for example,pharmaceutical agent, diagnostic agent, or an agent of nutritionalvalue.

Suitable pharmaceutical agents include, but are not limited to,anticancer or antineoplastic agents, antimicrotubule agents,immunosuppressive agents, anesthetics, hormones, agents for use incardiovascular disorders, antiarrhythmics, antibiotics, antifungals,antihypertensives, antiasthmatics, anti-inflammatory agents,anti-arthritic agents, vasoactive agents, analgesics/antipyretics,antidepressants, antidiabetics, antifungal agents, anti-inflammatories,antianxiety agents, immunosuppressive agents, antimigraine agents,sedatives, antianginal agents, antipsychotic agents, antimanic agents,antiarthritic agents, antigout agents, anticoagulants, thrombolyticagents, antifibrinolytic agents, hemorheologic agents, antiplateletagents, anticonvulsants, antiparkinson agents,antihistamines/antipruritics, agents useful for calcium regulation,antiviral agents, antimicrobials, anti-infectives, bronchodialators,hormones, hypoglycemic agents, hypolipidemic agents,antiulcer/antireflux agents, antinauseants/antiemetics, and oil-solublevitamins (e.g., vitamins A, D, E, K, and the like).

In some embodiments, the substantially water insoluble pharmacologicallyactive agent is any one of the following: a tyrosine kinase inhibitor, aseries/threonine kinase inhibitor, a hedgehog inhibitor, a topoisomeraseinhibitor, an in inhibitor of microtubule assembly, an inhibitor of theAKT kinase pathway, a proteasome inhibitor, an antimetabolite, and aplatinum-based agent.

In some embodiments, the substantially water insoluble pharmacologicallyactive agent is an antineoplastic agent. In some embodiments, thesubstantially water insoluble pharmacologically active agent is achemotherapeutic agent.

Suitable substantially water insoluble pharmacologically active agentsinclude, but are not limited to, taxanes (such as paclitaxel, docetaxel,ortataxel and other taxanes), epothilones, camptothecins, colchicines,geladanamycins, amiodarones, thyroid hormones, amphotericin,corticosteroids, propofol, melatonin, cyclosporine, rapamycin(sirolimus) and derivatives, tacrolimus, mycophenolic acids, ifosfamide,vinorelbine, vancomycin, gemcitabine, SU5416, thiotepa, bleomycin,diagnostic radiocontrast agents, and derivatives thereof. Othersubstantially water insoluble pharmacologically active agents that areuseful in the inventive compositions are described in, for example, U.S.Pat. Nos. 5,916,596, 6,096,331, 6,749,868, and 6,537,539. Additionalexamples of substantially water insoluble pharmacologically activeagents include those compounds which are poorly water soluble and whichare listed in the “Therapeutic Category and Biological Activity Index”of The Merck Index (12^(th) Edition, 1996).

In some embodiments, the substantially water insoluble pharmacologicallyactive agent is any of (and in some embodiments selected from the groupconsisting of) paclitaxel, docetaxel, CY196, ortataxel or other taxaneor taxane analog, 17-allyl amino geldanamycin (17-AAG), 18-derivatizedgeldanamycin, camptothecin, propofol, amiodarone, cyclosporine,epothilone, radicicol, combretastatin, rapamycin, amphotericin,liothyronine, epothilone, colchicine, thiocolchicine and its dimers,thyroid hormone, vasoactive intestinal peptide, corticosteroids,melatonin, tacrolimus, mycophenolic acids, epothilones, radicicols,combretastatins, and analog or derivative thereof. In some embodiments,the substantially water insoluble pharmacologically active agent is anyof (and in some embodiments selected from the group consisting of)paclitaxel, docetaxel, CY196, ortataxel or other taxanes, geldanamycin,17-allyl amino geldanamycin, thiocolchicine and its dimers, rapamycin,cyclosporine, epothilone, radicicol, and combretastatin. In someembodiments, the substantially water insoluble pharmacologically activeagent is rapamycin. In some embodiments, the substantially waterinsoluble pharmacologically active agent is 17-AAG. In some embodiments,the substantially water insoluble pharmacologically active agent is athiocolchicine dimer (such as IDN5404). In some embodiments, thesubstantially water insoluble pharmacologically active agent is ataxane. In some embodiments, the substantially water insolublepharmacologically active agent is paclitaxel. In some embodiments, thesubstantially water insoluble pharmacologically active agent isdocetaxel. In some embodiments, the substantially water insolublepharmacologically active agent is CY196.

In some embodiments, the substantially water insoluble pharmacologicallyactive agent is a taxane or derivative thereof, which includes, but isnot limited to, paclitaxel, docetaxel and IDN5109 (ortataxel), or aderivative thereof. In some embodiments, the composition comprises anon-crystalline and/or amorphous taxane (such as paclitaxel or aderivative thereof). In some embodiments, the composition is prepared byusing an anhydrous taxane (such as anhydrous docetaxel or a derivativethereof). Anhydrous docetaxel has been shown to produce more stableformulation than can be made with a hydrated docetaxel such as docetaxeltrihydrate or hemi-hydrate.

Other Components in the Nanoparticle Compositions

The nanoparticles described herein can be present in a composition thatincludes other agents, excipients, or stabilizers. For example, toincrease stability by increasing the negative zeta potential ofnanoparticles, certain negatively charged components may be added. Suchnegatively charged components include, but are not limited to bile saltsof bile acids consisting of glycocholic acid, cholic acid,chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid,taurochenodeoxycholic acid, litocholic acid, ursodeoxycholic acid,dehydrocholic acid and others; phospholipids including lecithin (eggyolk) based phospholipids which include the followingphosphatidylcholines: palmitoyloleoylphosphatidylcholine,palmitoyllinoleoylphosphatidylcholine,stearoyllinoleoylphosphatidylcholine stearoyloleoylphosphatidylcholine,stearoylarachidoylphosphatidylcholine, anddipalmitoylphosphatidylcholine. Other phospholipids includingL-α-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine(DOPC), distearyolphosphatidylcholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), and other related compounds. Negativelycharged surfactants or emulsifiers are also suitable as additives, e.g.,sodium cholesteryl sulfate and the like.

In some embodiments, the composition is suitable for administration to ahuman. In some embodiments, the composition is suitable foradministration to a mammal such as, in the veterinary context, domesticpets and agricultural animals. There are a wide variety of suitableformulations of the nanoparticle composition (see, e.g., U.S. Pat. Nos.5,916,596 and 6,096,331). The following formulations and methods aremerely exemplary and are in no way limiting. Formulations suitable fororal administration can consist of (a) liquid solutions, such as aneffective amount of the compound dissolved in diluents, such as water,saline, or orange juice, (b) capsules, sachets or tablets, eachcontaining a predetermined amount of the active ingredient, as solids orgranules, (c) suspensions in an appropriate liquid, and (d) suitableemulsions. Tablet forms can include one or more of lactose, mannitol,corn starch, potato starch, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, moistening agents, preservatives, flavoring agents,and pharmacologically compatible excipients. Lozenge forms can comprisethe active ingredient in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

Examples of suitable carriers, excipients, and diluents include, but arenot limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate, and mineral oil. Theformulations can additionally include lubricating agents, wettingagents, emulsifying and suspending agents, preserving agents, sweeteningagents or flavoring agents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation compatible with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Injectable formulations are preferred.

In some embodiments, the composition is formulated to have a pH range ofabout 4.5 to about 9.0, including for example pH ranges of any of about5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. Insome embodiments, the pH of the composition is formulated to no lessthan about 6, including for example no less than about any of 6.5, 7, or8 (such as about 8). The composition can also be made to be isotonicwith blood by the addition of a suitable tonicity modifier, such asglycerol.

Method of Using Prion-Free Nanoparticle Compositions

Also provided are methods of using the prion-free compositions describedherein. For example, in some embodiments, there is provided a method ofadministering a composition comprising nanoparticles comprising albuminand a substantially water insoluble pharmacologically active agent,wherein the composition is substantially free of a prion protein. Insome embodiments, there is provided a method of treating a disease (suchas cancer) comprising administering an effective amount of a compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, wherein the composition issubstantially free of a prion protein.

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe albumin in the composition was obtained by a method comprising aprion-removal process, said prion-removal process comprising contactingan initial albumin composition with a ligand capable of binding to aprion protein. In some embodiments, the prion removal process furthercomprises removing said ligand and proteins bound thereto from saidalbumin composition. In some embodiments, there is provided a method oftreating a disease (such as cancer) in an individual comprisingadministering to the individual an effective amount of compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, wherein the albumin in thecomposition was obtained by a method comprising a prion-removal process,said prion removal process comprising contacting an initial albumincomposition with a ligand capable of binding to a prion protein.

In some embodiments, the individual has vCJD. In some embodiments, theindividual has already been infected with a prion protein. In someembodiments, the individual is suspected of having vCJD or beinginfected with a prion protein. In some embodiments, the individual is anasymptomic carrier of a prion protein. In some embodiments, theindividual has received blood transfusion at least once. In someembodiments, the individual is at least 60 years old, such as at leastabout 65, 70, or 75 years old. In some embodiments, the individual isimmunity compromised. In some embodiments, the individual is a cancerpatient.

The term “effective amount” used herein refers to an amount of acompound or composition sufficient to treat a specified disorder,condition or disease such as ameliorate, palliate, lessen, and/or delayone or more of its symptoms. In reference to cancers or other unwantedcell proliferation, an effective amount comprises an amount sufficientto cause a tumor to shrink and/or to decrease the growth rate of thetumor (such as to suppress tumor growth). In some embodiments, aneffective amount is an amount sufficient to delay development. In someembodiments, an effective amount is an amount sufficient to preventoccurrence and/or recurrence. An effective amount can be administered inone or more administrations.

Cancers to be treated by compositions described herein (such as acomposition comprising an antineoplastic agent such as taxane,rapamycin, and 17-AAG) include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia. Examples of cancers that canbe treated by compositions described herein include, but are not limitedto, squamous cell cancer, lung cancer (including small cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung, including squamous NSCLC), cancer of theperitoneum, hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer (such as advanced pancreaticcancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer(such as hepatocellular carcinoma), bladder cancer, heptoma, breastcancer, colon cancer, melanoma, endometrical or uterine carcinoma,salivary gland carcinoma, kidney or renal cancer, liver cancer, prostatecancer (such as advanced prostate cancer), vulval cancer, thyroidcancer, hepatic carcinoma, head and neck cancer, colorectal cancer,rectal cancer, soft-tissue sarcoma, Kaposi's sarcoma, B-cell lymphoma(including low grade/follicular non-Hodgkin's lymphoma (NHL), smalllymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediategrade diffuse NHL, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL, bulky diseaseNHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom'smacroglobulinemia), chronic lymphocytic leukemia (CLL), acutelymphoblastic leukemia (ALL), myeloma, Hairy cell leukemia, chronicmyeloblastic leukemia, and post-transplant lymphoproliferative disorder(PTLD), as well as abnormal vascular proliferation associated withphakomatoses, edema (such as that associated with brain tumors), andMeigs' syndrome. In some embodiments, there is provided a method oftreating metastatic cancer (that is, cancer that has metastasized fromthe primary tumor). In some embodiments, there is provided a method ofreducing cell proliferation and/or cell migration. In some embodiments,there is provided a method of treating hyperplasia.

In some embodiments, there are provided methods of treating cancer atadvanced stage(s). In some embodiments, there are provided methods oftreating breast cancer (which may be HER2 positive or HER2 negative),including, for example, advanced breast cancer, stage IV breast cancer,locally advanced breast cancer, and metastatic breast cancer. In someembodiments, the cancer is lung cancer, including, for example,non-small cell lung cancer (NSCLC, such as advanced NSCLC), small celllung cancer (SCLC, such as advanced SCLC), and advanced solid tumormalignancy in the lung. In some embodiments, the cancer is ovariancancer, head and neck cancer, gastric malignancies, melanoma (includingmetastatic melanoma), colorectal cancer, pancreatic cancer, and solidtumors (such as advanced solid tumors). In some embodiments, the canceris any of (and in some embodiments selected from the group consistingof) breast cancer, colorectal cancer, rectal cancer, non-small cell lungcancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer,liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, gliomas, glioblastomas, neuroblastomas, and multiplemyeloma. In some embodiments, the cancer is a solid tumor.

Individual suitable for receiving these compositions depend on thenature of the poorly water soluble pharmaceutical agent, as well as thedisease/condition/disorder to be treated and/or prevented. Accordingly,the term individual includes any of vertebrates, mammals, and humans. Insome embodiments, the individual is a mammal, including, but not limitedto, human, bovine, equine, feline, canine, rodent, or primate. In someembodiments, the individual is human.

The dose of the inventive composition administered to an individual(such as human) will vary with the particular composition, the method ofadministration, and the particular disease being treated. The doseshould be sufficient to effect a desirable response, such as atherapeutic or prophylactic response against a particular disease. Forexample, the dosage of paclitaxel in the composition can be in the rangeof 100-400 mg/m² when given on a 3 week schedule, or 50-250 mg/m² whengiven on a weekly schedule. In addition, if given in a metronomicregimen (e.g., daily or a few times per week), the dosage may be in therange of about 5-75 mg/m².

The compositions described herein can be administered to an individual(such as human) via various routes, including, for example, intravenous,intra-arterial, intrapulmonary, intraportal, intrahepatic, oral,inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, transmucosal, and transdermal. For example,the inventive composition can be administered by inhalation to treatconditions of the respiratory tract. The composition can be used totreat respiratory conditions such as pulmonary fibrosis, broncheolitisobliterans, lung cancer, bronchoalveolar carcinoma, and the like.

In some embodiments, the administration of the composition is conductedin conjunction with a prion-removal filter.

Also provided herein are methods of reducing side effects associatedwith administration of the nanoparticle composition. For example, theinvention provides methods of reducing various side effects associatedwith administration of the poorly water soluble pharmaceutical agent,including, but not limited to, myelosuppression, neurotoxicity,hypersensitivity, inflammation, venous irritation, phlebitis, pain, skinirritation, peripheral neuropathy, neutropenic fever, anaphylacticreaction, hematologic toxicity, and cerebral or neurologic toxicity, andcombinations thereof. In some embodiments, there is provided a method ofreducing hypersensitivity reactions associated with administration ofthe poorly water soluble pharmaceutical agent, including, for example,severe skin rashes, hives, flushing, dyspnea, tachycardia, and others.

Kits and Systems

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprising the prion-freenanoparticle compositions, and in some embodiments, further compriseinstructions for use in accordance with any of the methods describedherein. The kit may further comprise a description of selection anindividual suitable or treatment. Instructions supplied in the kits ofthe invention are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable.

The kits of the invention are in suitable packaging. Suitable packaginginclude, but is not limited to, vials, bottles, jars, flexible packaging(e.g., seled Mylar or plastic bags), and the like. Kits may optionallyprovide additional components such as buffers and interpretativeinformation.

The instructions relating to the use of the nanoparticle compositionsgenerally include information as to dosage, dosing schedule, and routeof administration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Forexample, kits may be provided that contain sufficient dosages of thesubstantially water insoluble pharmacologically active agent (such assubstantially water insoluble pharmacologically active agent) asdisclosed herein to provide effective treatment of an individual for anextended period, such as any of a week, 2 weeks, 3 weeks, 4 weeks, 6weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9months, or more. Kits may also include multiple unit doses of thesubstantially water insoluble pharmacologically active agent andpharmaceutical compositions and instructions for use and packaged inquantities sufficient for storage and use in pharmacies, for example,hospital pharmacies and compounding pharmacies.

In some embodiments, there is provided a kit for removing a prionprotein from a nanoparticle composition comprising albumin andsubstantially water insoluble pharmacologically active agent, comprisinga ligand capable of binding to a prion protein. In some embodiments, thekit further comprises a supporting material. In some embodiments, thekit further comprises an instruction for using the ligand for removingprion from the nanoparticle composition.

Also provided are systems for carrying out methods described herein. Forexample, in some embodiments, there is provided a system formanufacturing prion-free nanoparticle composition comprising albumin anda substantially water insoluble pharmacologically active agent, saidsystem comprising 1) an apparatus for making the nanoparticlecomposition; and 2) an apparatus for removing prion proteins from thealbumin used for making the nanoparticle composition. In someembodiments, the apparatus for removing prion proteins from said albuminused for making the nanoparticle composition is integrated into theapparatus for making the nanoparticle composition. In some embodiments,the apparatus for making the nanoparticle composition is separated fromthe apparatus for removing the prion proteins from the albumin used formaking the nanoparticle composition.

in some embodiments, there is provided a system for manufacturingprion-free nanoparticle composition comprising albumin and asubstantially water insoluble pharmacologically active agent, saidsystem comprising 1) an apparatus for making the nanoparticlecomposition; and 2) an apparatus for removing prion proteins (forexample from an intermediate composition generated during the making ofthe nanoparticles or from the generated nanoparticle compositions). Insome embodiments, the apparatus for removing prion proteins isintegrated into the apparatus for making the nanoparticle composition.In some embodiments, the apparatus for making the nanoparticlecomposition is separated from the apparatus for removing the prionproteins.

Those skilled in the art will recognize that several embodiments arepossible within the scope and spirit of this invention.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Embodiments of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such embodiments asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible embodiments thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Exemplary Embodiments of the Present Application

In one aspect, there is provided a composition comprising nanoparticlescomprising albumin and a substantially water insoluble pharmacologicallyactive agent, wherein the composition is substantially free of a prionprotein. In some embodiments, the composition has a prion infectivity ofless than about 100 fg/ml. In some embodiments, the composition is anyone of the compositions described above, wherein the composition has aprion infectivity of less than about 10 IU-ic/ml. In some embodiments,the composition is any one of the compositions described above, whereinthe composition does not show the presence of a prion protein based on aprotein misfolding cyclic amplification (PMCA) assay or based on an IPCRassay. In some embodiments, the composition is any one of thecompositions described above, further comprising a trace amount of aligand capable of binding to a prion protein. In some embodiments, thecomposition is any one of the compositions described above, furthercomprising a trace amount of a supporting material.

In another aspect, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, wherein the albumin in the compositionwas obtained by a method comprising a prion removal process, said prionremoval process comprising contacting an initial albumin compositionwith a ligand capable of binding to a prion protein. In someembodiments, the prion removal process further comprises removing saidligand and proteins bound thereto from said albumin and composition. Insome embodiments, the ligand is a peptide. In some embodiments, theligand is a trazine-based compound.

In another aspect, there is provided a method of producing a compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, said method comprising: a)removing a prion protein from an initial albumin composition; b)subjecting a mixture comprising a solution comprising the prion-removedalbumin and an organic phase comprising said substantially waterinsoluble pharmacologically active agent dispersed in an organic solventto a high shear condition. In some embodiments, step a) comprises: 1)contacting the initial albumin solution with a ligand capable of bindingto a prion protein. In some embodiments, step a) further comprises: 2)removing the ligand and proteins bound thereto from the albuminsolution. In some embodiments, the method is any of the methodsdescribed above, further comprising removing said organic solvent fromthe mixture. In some embodiments, said removing of the organic solventis by evaporation. In some embodiments, the method is any one of themethods described above, wherein said ligand is a peptide. In someembodiments, the method is any one of the methods described above,wherein the ligand is a triazine-based compound. In some embodiments,the method is any one of the methods described above, wherein theinitial albumin composition is a blood derived product.

In another aspect, there is provided a method of producing a compositioncomprising nanoparticles comprising albumin and a substantially waterinsoluble pharmacologically active agent, comprising: a) subjecting amixture comprising an organic phase comprising said substantially waterinsoluble pharmacologically active agent and an albumin solution to ahigh shear condition, and b) removing a prion protein from said mixture.In some embodiments, step b) comprises: 1) contacting the mixture with aligand capable of binding to a prion protein. In some embodiments, stepb) further comprises: 2) removing the ligand and proteins bound theretofrom said mixture. In some embodiments, the ligand is a peptide. In someembodiments, the ligand is a triazine-based compound.

Also provided are composition produced by a method of any one of claims11-23. Also provided are uses of any one of the compositions describedabove for treating a disease, such as cancer.

In another aspect, there is provide a method of removing a prion proteinfrom a composition suspected of containing a prion protein comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, comprising: a) contacting thecomposition with a ligand capable of binding to a prion protein, b)removing the ligand and proteins bound thereto from the composition. Insome embodiments, the ligand is a peptide. In some embodiments, theligand is a triazine-based compound. Also provided are compositionsobtained after the method.

In another aspect, there is provided a composition comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, further comprising a ligand capable ofbinding to a prion protein. In some embodiments, the ligand is apeptide. In some embodiments, the ligand is a triazine-based compound.

The following examples are provided to illustrate, but not to limit, theinvention. It is understood that the examples described herein are forillustrative purposes only and that various modifications or changes inlight thereof will be suggested to persons skilled in the art and are tobe included within the spirit and purview of this application.

Examples Example 1 Development of an Affinity Adsorbent for Removal ofPrion Protein from Albumin Preparations

This study screened a four-resin panel of prion binding ligands bychallenging the four-resin panel with two different commerciallyavailable albumin preparations (containing 20% w/v or 25% w/v albumin),spiked with scrapie hamster brain homogenate at two differentconcentrations (0.01% or 0.005%). The four-resin panel was previouslyidentified by PRDT (Pathogen Removal and Diagnostic Technologies Inc;ProMetic Biosciences Ltd., Cambridge, UK) as good prion binders in thepresence of 25% albumin Selection of an optimum resin can optimize theincorporation of a prion-reduction step in the production of albuminnanoparticles.

Methodology

Six Protein Isolation Kit for Sorbent Identification (PIKSI™, ProMeticBiosciences Ltd) kits were packed with twelve columns (at about 0.5 mL)of each of the four PRDT resins and the control resin (Toyopearl AminoAMN31). Each resin was challenged with solutions containing 20% and 25%albumin spiked with 0.01% or 0.005% scrapie hamster brain homogenate(SBH) in a three-column series format in an effort to evaluate thebinding capacity of each resin to prion proteins. Comparison of resinperformance was based on prion protein binding as determined by Westernblot and densitometry. Total protein binding profile was determined bySDS-PAGE gels. The bound proteins were stripped from the resins for boththe prion protein binding and the total protein binding detection.Albumin binding was determined using NanoDrop® ND-1000 spectrophotometerto measure absorbance at 280 nm. The signals observed in the Westernblots and SDS-PAGE gels correspond to the bound fraction of prionprotein and total protein, respectively.

The commercial albumin preparations used in this invention were Albumin(Human) U.S.P. Human Albumin Grifols® 20%, Lot No. IBAB8MJ001, andAlbumin (Human), USP, 25% solution Baxter, Lot No. LA06D04AA.

Results Western Blot and SDS-PAGE

The results obtained show that all four resins bound PrPsc spiked into20% or 25% human albumin solutions. The PrPsc signal intensity inWestern blot (FIGS. 1-3) suggests that prion binding was strongest forDVR, followed by YVHEA, SYA, and D4 resins. The control resin, AMN31,had an expected strong signal. The level of signal obtained by using DVRresin suggests that high concentrations of albumin did not interfere inprion binding.

No detectable signal was observed in the second and third columns whenusing DVR (FIG. 1) at various conditions tested, even when longerexposure times were tested, which indicates that all the detectablePrPsc was captured by the first DVR column.

Signal intensity was weaker for the other resins in the first column,with detection of prion protein in the second column of the series(FIGS. 2 and 3), suggesting a possible interference of albumin to prionprotein binding. All three ligands showed no prion signal in the thirdcolumn, indicating that the resins are capable of selectively removingprions from the SBH spiked albumin solutions. The concentration of PrPscin hamster brain was about 50 μg/g, equivalent to about 5 ng/mL of PrPscin 0.01% SBII, spiked into a 250 mg/mL albumin solution, generating a50,000,000-fold excess of albumin

The total protein pattern observed in the Coomassie-stained SDS-PAGEgels (FIGS. 1 to 3) shows that DVR had a much lower level of totalprotein binding than the remaining prion-binding resins, which isconsidered an advantage, despite the fact that most of the observedbands in the total protein gel came from the brain homogenate spike. Asexpected, the one visible protein band in the DVR gel has an apparentmolecular weight similar to albumin (66.5 KDa).

Densitometry

Prion removal was also assessed indirectly by calculating the ratio ofdensitometric signal of PrPsc bound to the resin versus the signalpresent in the albumin solutions spiked with SBH. The ability of DVRresin to bind prion was comparably to the positive control resin,adsorbing approximately all available PrPsc in the first column Usinginfectious doses as the measurement for prion binding, the 0.5 mL DVRcolumn was able to remove prion in the 10 mL SBH-spiked albuminsolutions, which is equivalent to about 10⁶ ID₅₀, considering that 0.1%SBH contains about 10⁶ ID₅₀/mL. Similarly to what was observed in theWestern blots, DVR had the best performance in prion binding than YVHEA,D4, and SYA. All of three resins required the second column of eachseries to further bind any detectable prion protein present inSBH-spiked albumin solutions.

Albumin Binding

Albumin concentration was determined using a NanoDrop® ND-1000spectrophotometer at absorbance 280 nm for protein quantitation. Eachflow-through was measured for the concentration of albumin after passingthe SBH-spiked albumin solutions through each of the four resins (DVR,YVHEA, SYA, and D4), and the control resin (AMN31). The proteinconcentration of commercial albumin solutions at 20% and 25% in theabsence or presence of 0.01% or 0.005% SBH spikes was measured beforeflowing through the resin columns The results obtained are shown inTable 3.

TABLE 3 Measurement of albumin concentration of commercial albuminsolutions (20% w/v or 25% w/v) spiked with or without 0.01% and 0.005%scrapie hamster brain homogenate. Measured Measured ConcentrationConcentration Average (%) (mg/mL) (%) 20% albumin 24.8 248.0 24.6 20%albumin + 0.01% spike 24.7 247.1 20% albumin + 0.005% spike 24.3 243.325% albumin 26.8 268.1 27.6 25% albumin + 0.01% spike 28.3 282.5 25%albumin + 0.005% spike 27.8 278.3

The concentrations of the commercial albumin solutions were measuredhigher than the commercially labeled value, especially for thepreparation containing the 20% w/v albumin solution. However, since thestudy dealt with comparative values, this was not a concern. The averageof three values was obtained, and the amount of the spike was considerednegligible when compared to the amount of albumin present in thesolutions in determine protein concentration. The concentration ofalbumin was obtained after flowing SBH-spiked albumin solutions throughthe resins as shown in Tables 4 and 5.

TABLE 4 Measurement of albumin concentration after flowing commercialalbumin solution (20% w/v) spiked with or without 0.01% and 0.005%scrapie hamster brain homogenate through different resin columns.Concentration Albumin (mg/mL) Loss (%) DVR  0.01% spike 260.4 ND 0.005%spike 260.4 ND YVHEA  0.01% spike 264.3 ND 0.005% spike 259.5 ND SYA 0.01% spike 245.5 0.2 0.005% spike 241.7 1.7 D4  0.01% spike 263.0 ND0.005% spike 265.8 ND AMN31  0.01% spike 249.8 ND 0.005% spike 246.9 NDND = not detected.

TABLE 5 Measurement of albumin concentration after flowing commercialalbumin solution (25% w/v) spiked with or without 0.01% and 0.005%scrapie hamster brain homogenate through four different resin columns.Concentration Albumin (mg/mL) Loss (%) DVR  0.01% spike 268.8 2.6 0.005%spike 284.2 ND YVHEA  0.01% spike 285.7 ND 0.005% spike 294.2 ND SYA 0.01% spike 280.5 ND 0.005% spike 262.9 4.7 D4  0.01% spike 284.0 ND0.005% spike 261.1 5.4 AMN31  0.01% spike 272.4 1.3 0.005% spike 282.4ND ND = not detected.

None of the resins tested showed a significant loss of albumin In fact,no loss was detected for most of the conditions. The values foundindicate a variation of about ±5%. The data suggest that albumin loss isunlikely a factor for choosing one of the resins at this scale, withinthe range of tested conditions.

Conclusions

Based on the results obtained, DVR is the best resin among the fourtested resins for removing prion from commercial albumin solutions. Theresin was able to remove around 10⁶ ID₅₀ in a 0.5-mL column. This valueis similar to the ones obtained in previous trials for other challenges.There was little loss of albumin detected at the tested ratio ofchallenge with 20 or 25% albumin/resin of 20. Albumin binding should belower at process conditions, since this ratio is likely to increase.

Example 2 Prion-Removal Feasibility Study of Formulated SuspensionsContaining Paclitaxel and of Human Albumin Solution

Application of the prion-removal technology in the nanoparticlecomposition was evaluated using formulated suspensions containingpaclitaxel (e.g., Abraxane™) and using human albumin solution containingvarious percentages of albumin (% w/v).

Experimental Conditions Study Systems

Formulated Suspensions for Abraxane™ and sugar-paclitaxel formulationwere evaluated. Formulated Suspension (FS) for Abraxane™ was formulatedwith post-evaporated suspension (PE) obtained from the production andhuman albumin solution (25%, Baxter, Deerfield, Ill.), containingapproximately 7 mg/mL paclitaxel and 56 mg/mL human albumin FormulatedSuspension (FS) for sugar-paclitaxel formulation was formulated withpost-evaporated suspension (PE) obtained from the production, humanalbumin solution (25%, Baxter), sucrose, sodium chloride and edetatedisodium dihydrate, containing approximately 7 mg/ml paclitaxel, 56mg/ml human albumin; 32 mg/ml sucrose, 8.4 NaCl mg/ml, and 0.07 mg/mlEDTA (ethylenediaminetetraacetic acid).

Human Albumin Solutions in 25%, 20%, and 5% were also evaluated. Humanalbumin solution in 25% or 250 mg/ml was obtained from Baxter; humanalbumin solution in 20% or 200 mg/ml (e.g., Grifols®) was obtained fromGrifols Biologicals, Inc. (Los Angeles, Calif.). Human albumin solution5% was made by dilution of 25% human albumin (Baxter) with sterile waterfor injection.

Prion-Removal Columns

The prion removal columns (1-ml) used in this study were commerciallyavailable PIKSI® (Protein isolation Kit for Sorbent Identification) kitcolumns containing Toyopearl Amino 650CU resin (sample AMN31) suppliedby ProMetic Biosciences, Ltd (Cambridge, UK).

Flow-Through Conditions

For the study on formulation suspension, the total flow-through volumewas about 500 mL, which was 500 times the column volume (1-ml). The flowrate was about 0.5 ml/min Total flow-through time was more than 16hours. Samples were taken every two hours. No clogging of the column wasobserved.

For the study on albumin solution, the total flow-through volume was atleast 100 mL, which was 100 times the column volume (1-ml). The flowrate was about 0.5 ml/min Total flow-through time was more than 3 hours.Samples were taken every hour. No clogging of the column was observed.

Results and Discussion

The results from the prion-removal feasibility study on the formulatedsuspension are summarized in Table 6 and Table 7. The physical andchemical testing results for the formulated suspension for Abraxane™show no significant differences between the pre and post-columnsuspension, in terms of particle size, pH, paclitaxel assay andimpurities, human albumin assay, and human albumin composition.Likewise, the physical and chemical testing results for the formulatedsuspension for sugar-paclitaxel formulation show no significantdifferences between the pre and post-column suspension, in terms ofparticle size, pH, paclitaxel assay and impurities, human albumin assay,human albumin composition, sucrose, and EDTA.

TABLE 6 Prion-Removal Feasibility Study of Abraxane ™ FormulatedSuspension Sample I.D. Physical and Chemical Pre- Post Column PropertiesColumn #1 #2 #3 #4 #5 #6 #7 Particle Size Mean 131 132 132 133 133 133131 130 (nm)   <5% 82 86 85 86 85 87 84 82   <95% 193 190 191 192 192191 191 190 <99.9% 254 250 251 252 253 250 251 250 pH 7.0 7.0 7.1 7.07.0 7.0 7.0 7.0 Paclitaxel (mg/mL) 6.9 6.9 6.9 6.8 6.8 6.8 6.8 6.8 HumanPolymer 3.99 3.86 3.93 3.95 3.95 3.92 3.88 3.76 Albumin Oligomer 1.321.36 1.32 1.34 1.31 1.32 1.34 1.39 Composition Dimer 5.95 5.96 6.00 5.986.00 5.99 5.98 5.96 (%) Monomer 88.29 88.37 88.24 88.27 88.27 88.2888.29 88.38 Total Human Albumin 55 56 56 56 56 56 56 56 (mg/mL) Impurity7-Epi 0.08 0.08 NA NA 0.09 NA NA 0.09 (%) Total 0.25 0.25 NA NA 0.26 NANA 0.26 * NA: Data not Available.

TABLE 7 Prion-Removal Feasibility Study of Sugar-EDTA-PaclitaxelFormulated Suspension Sample I.D. Physical and Chemical Pre- Post ColumnProperties Column #1 #2 #3 #4 #5 #6 #7 Particle Size Mean 129 130 133131 132 132 130 132 (nm)   <5% 80 81 85 82 84 85 81 85   <95% 191 192193 193 192 191 192 191 <99.9% 252 253 253 254 253 251 253 250 pH 6.86.8 6.8 6.9 6.8 6.8 6.9 6.9 Paclitaxel (mg/mL) 6.5 6.5 6.5 6.5 6.5 6.66.6 6.5 Human Polymer 3.90 3.90 3.91 3.89 3.91 3.93 3.94 3.93 AlbuminOligomer 1.92 1.89 1.92 1.90 1.91 1.89 1.44 1.41 Composition Dimer 6.146.11 6.11 6.10 6.10 6.09 6.09 6.10 (%) Monomer 88.02 88.10 88.07 88.0988.06 88.09 88.03 88.04 Total Human Albumin 55 55 54 54 55 55 54 54(mg/mL) Impurity 7-Epi 0.08 0.08 NA NA 0.08 NA NA 0.09 (%) Total 0.250.25 NA NA 0.25 NA NA 0.26 Sucrose (mg/mL) 31.8 31.8 NA NA 31.6 NA NA31.9 EDTA (mg/mL) 0.071 0.071 NA NA 0.070 NA NA 0.070 * NA: Data notAvailable.

The results from the prion-removal feasibility study on the albuminsolutions are summarized in Table 8. There are no significantdifferences between the pre and post-column solution, in terms of humanalbumin assay and human albumin composition.

TABLE 8 Prion-Removal Feasibility Study of Human Albumin Solution HA (%)HA Total Sample I.D.  Polymer Oligomer Dimer Monomer (mg/mL) Lot28203-37A 25% Pre column 3.66 0.28 2.38 93.69 248 Human Post column #13.60 0.25 2.41 93.74 248 Albumin Post column #2 3.63 0.28 2.38 93.71 250(Baxter) Post column #3 3.64 0.24 2.41 93.71 247 Post column #4 3.640.26 2.40 93.70 248 Lot 28203-37B 20% Pre column 4.85 0.51 3.25 91.39199 Human Post column #1 4.78 0.49 3.25 91.48 199 Albumin Post column #24.78 0.51 3.25 91.45 200 (Grifols) Post column #3 4.86 0.51 3.25 91.38200 Lot 28203-37C  5% Human Pre column 3.66 0.27 2.32 93.75 52 AlbuminPost column #1 3.33 0.25 2.31 94.10 51 (Baxter) Post column #2 3.48 0.272.30 93.94 51 Post column #3 3.54 0.27 2.31 93.89 51 Post column #4 3.500.27 2.31 93.92 52

This study demonstrates that the prion-removal column treatment has noadverse impact on the physical and chemical properties of human albuminsolution and formulated suspensions for both Abraxane™ andsugar-paclitaxel formulation.

Example 3 TSE Removal by Prion Reduction Resins for 20% Albumin

In this study, potential TSE removal by prion reduction resins (PRDTcolumn; ProMetic Biosciences, Ltd) in 20% (w/v) albumin (Grifols®) wasevaluated. Starting material for the TSE removal process step was spikedwith a model TSE agent. The process step was performed in the VirusSurelaboratories (Virusure Forschung und Entwicklung GmbH, Vienna, Austria).Various fractions were collected during performance of the process step,and the TSE removal capacity of the process was calculated based on adetermination of levels of TSE agent using a Western Blot assay for thedetection of PrPsc.

This study followed and referenced the following guidelines,including 1) CPMP/BWP/268/95 (revised in 1996), Note for Guidance onVirus Validation Studies: The Design, Contribution, and Interpretationof Studies Validating the Inactivation and Removal of Viruses; 2)CPMP/BWP/5136/03 Guideline on the Investigation of ManufacturingProcesses for Plasma-Derived Medicinal Products with Regards to vCJDRisk; 3) OECD Principles of Good Laboratory Practice as outlined inENV/MC/CHEM(98)17, revised in 1997; 4) 21 CFR part 58, Good LaboratoryPractice, US FDA; 5) EU directive 2004/9/EG, Inspection und Überprüfungder Guten Laborpraxis (GLP); 6) EU Directive 2004/10/EG, Anwendung derGrundsätze der guten Laborpraxis und zur Kontrolle ihrer Anwendung beiVersunchen mit chemischen Stoffen; 7) Austrian BGBI. II, 211.Verordnung, Chemikalien-GLP-Inspektionsverordnung, Jahrgang 2000; and 8)Austrian BGBI. II, 450. Verordnung, gute Laborpraxis 2006, Jahrgang2006.

Materials and Methods

The following test articles, reagents, and materials were used duringthe course of this study for the investigation of TSE removal by theprion reduction resins (PRDT column) for 20% albumin (Grifols®).

Grifols® Albumin (human) (USP 20% solution (Lot: IBAB7GX001/TA09/0122))was used for spiked run and interference testing.

Disposable SepFast™ Column (ProMetic Biosciences Ltd.) containing 5 mlof prion removal resin packed in 9% saline was used for process run.

Various buffers were prepared. They include the following:

-   1) 0.9% NaCL (9 g/l) was prepared as equilibration buffer for the    prion reduction resins;-   2) Tris Buffered Saline (TBS) was prepared as re-suspension of the    resins following chromatography;-   3) 2M NaCl (116.88 g/l) was prepared as prion reduction resin    regeneration buffer;-   4) NaOH (0.1 M, 0.5M, and 1.0 M) buffers were prepared for pH    adjustments of spiked study samples and process intermediates    inactivation of infectious material; and-   5) HCL (0.1 M and 1.0 M) buffers were prepared for pH adjustments of    spiked study samples and process intermediates.

263 Scrapie

Strain 263K Hamster Adapted scrapie (0.5% sarkosyl treated) was used inthis study. The 263K strain of hamster adapted scrapie provides theadvantages of high titres in the brains of hamsters. Typical titres fora 10% brain homogenate are in the range of 10⁸-10¹⁰ ID₅₀ units per ml.The PrPsc protein deposited by this agent is relatively resistant toProteinase K digestion, allowing the possibility of distinguishingbetween the non-disease associated form of the protein PrPc. The seed263K strain of hamster adapted scrapie was supplied as a 10% homogenateby the laboratory of Dr. Robert Rohwer (Baltimore Research and EducationFoundation, Mail Stop 151-A, 10 North Greene Street, Baltimore, Md.21201, USA). A 0.5% sarkosyl-treated fraction was selected for thisexperiment. This fraction was prepared from a crude brain homogenate(from which the microsomal/cytosolic 263K fraction had already beenremoved) by treatment with 0.5% sarkosyl followed by differentialcentrifugation to remove larger aggregates, leaving only the detergentsolubilized fragments in the supernatant. The 0.5% sarkosyl-treatedfraction was prepared to mimic detergent solubilized contamination(i.e., as found in solvent detergent containing processes), and thistype of fraction has been widely used in prion clearance studies forhuman plasma and recombinant products.

Western Blot Assay for the Detection of PrPsc

The Western blot assay for detection of PrPsc was used for thesemi-quantitative determination of TSE levels (PrPsc) in the varioussamples. The dynamic range of the Western blot assay is normally in theregion of 4-5 log₁₀ dilutions before signal is lost, and thus the assayis less sensitive than the hamster bioassay. However, the Western blotassay is a useful tool in assessing prion removal by biopharmaceuticalmanufacturing processes.

In the Western blot assay, the sample was first submitted to digestionwith Proteinase K to remove the normal form of the protein, PrPc (allprocess samples were digested using a Proteinase K concentration of 83pg/ml). After blocking of the proteolytic reaction, the sample was mixedwith SDS-buffer and boiled to denature the PrPsc from its aggregatedform. 0.5 log₁₀ dilutions of the sample are then prepared and loadedonto a SDS-PAGE gel along with a molecular weight marker. Followingelectrophoresis, the gel was Western blotted onto a PVDF membrane,followed by blocking and probing with antibodies allowing the detectionof bound PrPsc protein with the antibody 3F4. The 263K strain of scrapieresults in a characteristic banding pattern in the region of 25-33 KDa,which assists in confirming the presence of the PrPsc protein insamples. The end point of titre of the sample was defined as the firstdilution where no signal was observed on the Western blot.

Calculation of Reduction Factors

Reduction factors (RF) were calculated as follows:

RF=(V ₁ ×T ₁)/(V ₂ ×T ₂)

Where:

V₁ and T₁ are the volume and titre of the starting materialrespectively, and

V₂ and T₂ are the volume and titre of the product fraction respectively

In logarithmic terms, this equation can be expressed as:

Log₁₀[RF]=[ Log₁₀(V ₁)+Log₁₀(T ₁)]−[ Log₁₀(V ₂)+Log ₁₀(T ₂)].

Reduction factors were rounded to 1 decimal place only after the finalcalculation.

Interference Testing

The starting material was tested undiluted and followed by a 1.0 log₁₀pre-dilution in TBSA. Following spiking with 263K to a finalconcentration equivalent to a titre within approximately 2 log₁₀ of theend point of the Scrapie stock used for spiking, the undiluted andpre-diluted starting material samples were centrifuged at 15.558×g for60 min at room temperature. Following centrifugation, the supernatantwas carefully decanted and the pellets re-suspended with TBSA in 1/10 ofthe original centrifuged sample volume (equivalent to no effectiveconcentration for 1.0 log₁₀ pre-diluted sample and equivalent to a10-fold concentration for the undiluted sample). Protease K digestionand Western blotting was then performed following the standard protocol.The regeneration and column samples were diluted 0.5 log₁₀ or testedundiluted respectively prior to analysis by Western blotting (i.e.,without centrifugation).

Adjustment of pH

Prior to aliquotting and storage at ≦−60° C., samples were checked to beat pH 6-8 (pH adjustment was not required for any of the samples).

Equipment

The following main equipment items were used for performance of thisstudy. Sterile Class II Biohazard Safety Cabinet, Sanyo & Angelantoni≦−60° C. freezers, Angelantoni 2-8° C. fridges and ≦−15° C. freezers,Sartorius or Kern (analytical) balance and printer, Hanna electronicthermometer, Mettler Toledo pH meter, Grant or Selecta water baths,Oregon Laboratory Timer, Hettich microfuge, BioRad CriterionElectrophoresis Cell, BioRad Criterion Blotter, AKTA Chromatographysystem, Wealtec Power Supply, Agfa Film Developer, and Biotoolomicscolumn packed with PRDT resin.

Process Flow

The process flow scheme along with the samples collected was depicted inFIG. 4. The volumes of the respective samples can be found in Table 9 inthe Results Section.

In preparation of the process flow, the Laminar Flow (LF) safety cabinetwas cleaned and turned on for at least 10 to 15 minutes to equilibrate.The water bath was equilibrated to 30±2° C. and the starting materialequilibrated until a temperature of 29.5° C. was reached. The 0.5%Sarkosyl solubilized spike was thawed in the same water bath and as soonas it was thawed, placed on ice until use. The PRDT column wasequilibrated to ambient temperature (23.0±5.0° C.) overnight.

The column inlet tubing (top) was connected to the outlet from the ÄKTA.The tubing from the column outlet (bottom) was fed to an appropriatecollection vessel (beaker or 50 ml disposable centrifuge tube). Alltubing was already primed with WFI (Water for Injection) such that noair bubbles were introduced into the system.

The column was first equilibrated with 5 column volumes (CV) of WFI, andthen with 10 CV of Equilibration Buffer. The target flow rate throughoutwas 2.0±0.1 ml per minute.

Sample Preparation and Prion Reduction Resin Chromatography

50.7 ml of the equilibrated starting material was spiked with 0.51 ml of0.5% sarkosyl solubilized 263K homogenate. The pH was then be checkedand found to be within the target range of 6.9-7.4. Subsequently, a 0.5ml aliquot was removed (sample SSM) and aliquotted and stored at ≦−60°C.

The spiked sample was then applied to the above equilibrated PRDT columnat a flow rate of 1.8±0.1 ml per min and the flow through collected asthe following fractions:

Sample ID Volume Sample Description EI 2.1 ml Eluate 1 (−0-2 ml) E2 3.2ml Eluate 2 (−2-5 ml) E3 5.4 ml Eluate 3 (−5-10 ml) E4 16.1 ml  Eluate 4(−10-25 ml) E5 30.3 ml  Eluate 5 (−25-44 ml)

Collection of E1 began once the absorbance had reached 80% of the fullscale deflection. For each run, the flow through fraction was collected(the volume of each Eluate sample collected was determined by weighing)After loading of the spiked Albumin solution, the column was washed with10.0 ml of Equilibration Buffer. Collection of the E5 sample was stoppedonce the absorbance had dropped below 80% of full scale deflection.Following the Equilibration Buffer wash, the column was regeneratedusing >20.0 ml of 2M NaCl (sample REG), and following regeneration theresin removed and resuspended in 5 ml of TBS (sample COL).

Results

Samples from Spiked Runs

Table 9 below lists the samples that were collected from the spiked runalong with the volume of each sample. Where the sample size wasdetermined by weight, then a density of 1.0 g/ml was assumed to allow acalculation of the volume for each sample. All samples were storedaliquotted at −60° C. until analysis. A scanned reproduction of thechromatography profile from the spiked run is shown in FIG. 5.

TABLE 9 Summary of volumes collection during the process run Actualvolume of sample collected at Sample Description Sample Code point ofcollection (ml) Spiked Start Material SP0913-SSM 51.2 (Sarkosyl-treatedspike) Eluate 1: 0-2 ml SP0913-E1 2.1 Eluate 2: 2-5 ml SP0913-E2 3.2Eluate 3: 5-10 ml SP0913-E3 5.4 Eluate 4: 10-25 ml SP0913-E4 16.1 Eluate5: 25--44 ml SP0913-E5 30.3 Regeneration fraction SP0913-Reg 27.5 Resinsample SP0913-Col 10.0

Interference Results

To overcome interference all samples except the regeneration and resinsamples were diluted by 1.0 log₁₀ with TBS containing 0.1% BSA followedby a centrifugation and a 1/10 concentration. The undiluted sampletested for interference at a 10× concentration displayed stronginterference. For the regeneration samples a 0.5 log₁₀ dilution wasprepared prior to testing to reduce the concentration of NaCl. For theresin samples, as the resin was resuspended in TBS buffer, these sampleswere tested without pre-dilution.

The dilution of sample required to overcome interference with albuminwas made using 1.0 Log10 predilution with centrifugation andresuspension in 1/10^(th) original volume. See FIG. 6.

Prion Titration Data and Calculation of Reduction Factors

The calculation of the prion reduction factors for the process runs isshown in Table 10. The dilution of sample used in order to overcomeinterference is also shown in Table 10.

TABLE 10 Summary of Sample Titration Data and Prion Reduction FactorsLog Volume of dilution Sample Correction Volume of Samples pH Sample forEnd Volume Volume Factor for Samples After Adjustment Descriptioninterf- point at Further Volume Before pH pH Correction Log Total SampleID Spiked erence titre collection Processed processed* AdjustmentAdjustment Factor volume Load SP0913- Spiked start 0.0 2.5 51.2 ml 50.7ml 0.99 — 1.7 4.205 SSM material SP0913-E1 Eluate Fraction 1 0.0 0.050.7 ml — — 1.7 ≦1.705 SP0913-E2 Eluate Fraction 2 0.0 1.0 50.7 ml — —1.7 2.705 SP0913-E3 Eluate Fraction 3 0.0 1.0 50.7 ml — — 1.7 2.705SP0913-E4 Eluate Fraction 4 0.0 1.5 50.7 ml — — 1.7 3.205 SP0913-E5Eluate Fraction 5 0.0 1.0 50.7 ml — — 1.7 2.705 SP0913-Reg Regeneration0.5 0.0 27.5 ml — — 1.4 ≦1.939 Fraction SP0913-Col Column Resin 0.0 3.010.0 ml — — 1.0 4.000 *Correction factors are applied in the final logvolume calculation. The correction factors applicable for each sampleare the correction factors of the respective sample itself along withall correction factors for samples listed below that sample. **A volumeof 50 ml was used for the purpose of calculating the reduction factorfor each eluate sample, to ensure a direct comparison with the volumeloaded onto the column. Reduction Factor 1: Sample # Sample ID SampleDescription Log Titre RF Sample 1 SP0913-SSM Spiked start material 4.205Sample 2 SP0913-E1 Eluate Fraction 1 1.705 ≧2.50 Comments: RF for eluate1 relative to spiked start material Reduction Factor 2: Sample # SampleID Sample Description Log Titre RF Sample 1 SP0913-SSM Spiked startmaterial 4.205 Sample 6 SP0913-E5 Eluate Fraction 5 2.705   1.50Comments: RF for eluate 5 relative to spiked start material

A reduction factor of 2.5 log₁₀ relative to the spiked start materialwas calculated for E1 sample, which represented the first 2.0 ml passedover the PRDT column. In samples E2 to E5, a breakthrough of PrPsc intothe flow through fraction was observed (reduction factor 1.5 log₁₀ forsample E5). No PrPsc was detected in the regeneration sample and theequivalents of PrPsc bound to the PRDT resin was discussed in moredetail in the section of calculation of the prion binding capacity ofthe PRDT resin below.

Calculation of the Prion Binding Capacity of the Prion Reduction Resins

In order to estimate the binding capacity for the prion reduction resins(PRDT resin) for infectious prion protein, the titres observed in theWestern blot testing were related to infectious titres. The Western blotassay used a 263K stock of known titre. A dilution series of thereference stock was prepared and tested in the Western blot assay. Afterplotting the obtained titres versus the respective titres observed inthe hamster bioassay, a linear regression analysis was performed toassess the relation between the two test systems. The slope andintercept for the regression line were calculated to be 1.0667 and−4.5867, respectively. The regression parameters were used to convertWestern blot titres into infectious titres using the following formula:

Titre_([Bioassay])=(Titre_([WesternBlot])+4.5867)/(1.0667).

Once the infectious titre per ml was calculated, the total prion boundto the column could be determined. The calculated prion protein bindingcapacity of the prion reduction resin is shown in the Table 11 below.The capacity was determined using the amount of PrPsc observed directlybound to the prion reduction resin (samples Reg and Col).

TABLE 11 Prion binding capacity of Prion Reduction Resin (PRDT Resin)Binding capacity Infectious PrPsc per Sample ID Sample Description TotalPrPsc (log₁₀ ID₅₀ total) = ml Albumin spiked with a sarkosyl solubilisedspike (log₁₀ ^(WB))^(#) Total PrPsc + 4.1 (ID₅₀/ml)* SP0913-SSM 50 mlalbumin spiked with 0.5 ml 4 2

8.3 — of a sarkosyl solubilised spike SP0912-Reg 2M NaCl salt wash(sample ≦1.9 ≦6.0 ≦5.3 log₁₀/ml volume 25.1 ml) 5.0 ml PRDT Resin; 50 mlof SP0912-SSM sample loaded (Target flow rate: 1.8 ml/min) SP0912-ColColumn resin (sample volume 4.0 8.1   7.4 log₁₀/ml 8.7 ml) ^(#)TotalPrPSc includes corrections for volume of sample as well asconcentration/dilution prior to testing

Total load based on a final volume of 50 ml loaded onto the PRDT column*Based on a 5.0 ml column size

The total binding of PrPsc to the PRDT resin following a 2M salt washwas 7.4 log₁₀ ID₅₀/ml. No PrPsc was detected in the salt wash fractionsby western blot. Significant PrPsc removal (≧2.5 log₁₀) was alsoobserved.

When performing interference testing, the starting material was testedundiluted, with centrifugation and 10-fold concentration. This wasperformed to allow an evaluation of the possibility for concentratingthe samples and thus achieving higher reduction factors.

Example 4 TSE Removal by the Prion Reduction Resins for 25% Albumin

In this study, potential TSE removal by the prion reduction resins (PRDTcolumn) in 25% albumin (Baxter, Deerfield, Ill.) was evaluated.Materials and Methods used were the same as described in Example 3, withthe exception that Albumin (Human) USP 25% solution from Baxter (Lot:LA07D051AB/TA09/0123) was used as the starting material for spiked runand interference testing.

Process Flow

The downscale process was established and performed at the ViruSurefacilities. The process flow scheme along with the samples collected wasdepicted in FIG. 7. The volumes of the respective samples can be foundin Table 12 in the Results Section. The parameters and procedures usedin preparation of the process flow, including connecting the AKTA andcolumn, column equilibration are the same as described in Example 3.

Sample Preparation and Prion Reduction Resin Chromatography

50.1 ml of the equilibrated starting material was spiked with 0.51 ml of0.5% sarkosyl solubilized 263K homogenate. The pH was then be checkedand found to be within the target range of 6.9-7.4 with 0.1 M HCL or 0.5M NaOH. The pH of the spiked starting material (6.85) was outside thetarget range of 6.9-7.4 (see Deviations Section). After the addition of65 μL 0.1 M NaOH and 50 μl 1 M NaOH, the pH value remained unchanged, sothe decision was taken to load the starting material at this pH.Subsequently, a 0.5 ml aliquot was removed (sample SSM) and aliquottedand stored at ≦−60° C.

The spiked sample was then applied to the above equilibrated PRDT columnat a flow rate of 1.8±0.1 ml per min, and the flow through werecollected as the following fractions:

Sample ID Volume Sample Description EI 2.3 ml Eluate 1 (−0-2 ml) E2 3.3ml Eluate 2 (−2-5 ml) E3 5.6 ml Eluate 3 (−5-10 ml) E4 16.1 ml  Eluate 4(−10-25 ml) E5 32.7 ml  Eluate 5 (−25-44 ml)

Collection of E1 began once the absorbance had reached 80% of the fullscale deflection. For each run, the flow through fraction was collected(the volume of each Eluate sample collected was determined by weighing)After loading of the spiked albumin solution, the column was washed with10.0 ml of Equilibration Buffer. Collection of the E5 sample was stoppedonce the absorbance had dropped below 80% of full scale deflection.Following the Equilibration Buffer wash, the column was regeneratedusing >20.0 ml of 2M NaCl (sample REG), and following regeneration theresin removed and resuspended in 5 ml of TBS (sample COL).

Results

Samples from Spiked Runs

Table 12 below lists the samples that were collected from the spiked runalong with the volume of each sample. Where the sample size wasdetermined by weight, then a density of 1.0 g/ml was assumed to allow acalculation of the volume for each sample. All samples were storedaliquotted at ≦−60° C. until analysis. A scanned reproduction of thechromatography profile from the spiked run is shown in FIG. 8.

TABLE 12 Summary of volumes collection during the process run Actualvolume of sample collected at Sample Description Sample Code point ofcollection (ml) Spiked Start Material SP0912-SSM 50.6 (Sarkosyl-treatedspike) Eluate 1: 0-2 ml SP0912-E1 2.3 Eluate 2: 2-5 ml SP0912-E2 3.3Eluate 3: 5-10 ml SP0912-E3 5.6 Eluate 4: 10-25 ml SP0912-E4 16.1 Eluate5: 25--44 ml SP0912-E5 32.7 Regeneration fraction SP0912-Reg 25.1 Resinsample SP0912-Col 8.7

Interference Results

To overcome interference with albumin, all samples except theregeneration and resin samples were diluted by 1.0 log₁₀ with TBScontaining 0.1% BSA followed by a centrifugation and a 1/10concentration. The undiluted sample tested for interference at a 10-foldconcentration displayed strong interference. For the regenerationsamples, a 0.5 log₁₀ dilution was prepared prior to testing to reducethe concentration of NaCl. For the resin samples, as the resin wasresuspended in TBS buffer, these samples were tested withoutpre-dilution.

The dilution of sample required to overcome interference was made using1.0 Log10 predilution with centrifugation and resuspension in 1/10^(th)of the original volume. See FIG. 9.

Prion Titration Data and Calculation of Reduction Factors

The calculation of the prion reduction factors for the process runs isshown in Table 13. The dilution of sample used in order to overcomeinterference is also shown in Table 13.

TABLE 13 Summary of Sample Titration Data and Prion Reduction FactorsLog Volume of dilution Sample Correction Volume of Samples pH Sample forEnd Volume Volume Factor for Samples After Adjustment Descriptioninterf- point at Further Volume Before pH pH Correction Log Total SampleID Spiked erence titre collection Processed processed* AdjustmentAdjustment Factor volume Load SP0912- start material 0.0 2.0 50.6 ml50.2 ml 0.99 50.6 ml 50.7 ml 1.00 1.7 3.702 SSM SP0912-E1 EluateFraction 1 0.0 0.0 50.2 ml — — 1.7 ≦1.701 SP0912-E2 Eluate Fraction 20.0 0.5 50.2 ml — — 1.7 2.201 SP0912-E3 Eluate Fraction 3 0.0 1.0 50.2ml — — 1.7 2.701 SP0912-E4 Eluate Fraction 4 0.0 1.0 50.2 ml — — 1.72.701 SP0912-E5 Eluate Fraction 5 0.0 1.0 50.2 ml — — 1.7 2.701SP0912-Reg Regeneration 0.5 0.0 25.1 ml — — 1.4 ≦1.900 FractionSP0912-Col Column Resin 0.0 3.0 8.7 ml — — 0.9 3.940 *Correction factorsare applied in the final log volume calculation. The correction factorsapplicable for each sample are the correction factors of the respectivesample itself along with all correction factors for samples listed belowthat sample. **A volume of 50 ml was used for the purpose of calculatingthe reduction factor for each eluate sample to ensure a directcomparison with the volume loaded onto the column. Reduction Factor 1:Sample # Sample ID Sample Description Log Titre RF Sample 1 SP0912-SSMSpiked start material 3.702 Sample 2 SP0912-E1 Eluate Fraction 1 1.701≧2.00 Comments: RF for eluate 1 relative to spiked start materialReduction Factor 2: Sample # Sample ID Sample Description Log Titre RFSample 1 SP0912-SSM Spiked start material 3.702 Sample 6 SP0912-E5Eluate Fraction 5 2.701   1.00 Comments: RF for eluate 5 relative tospiked start material

A reduction factor of ≧2.5 log₁₀ relative to the spiked start materialwas calculated for E1 sample, which represented the first 2.0 ml passedover the PRDT column In samples E2 to E5, a breakthrough of PrPsc intothe flow through fraction was observed (reduction factor 1.5 log₁₀ forsample E5). No PrPsc was detected in the regeneration sample and theequivalents of PrPsc bound to the PRDT resin was discussed in moredetail in the section of calculation of the prion binding capacity ofthe PRDT resin below.

Calculation of the Prion Binding Capacity of the Prion Reduction Resins

In order to estimate the binding capacity for the prion reduction resins(PRDT resin) for infectious prion protein, the titres observed in theWestern blot testing were related to infectious titres. The Western blotassay used a 263K stock of known titre. A dilution series of thereference stock was prepared and tested in the Western blot assay. Afterplotting the obtained titres versus the respective titres observed inthe hamster bioassay, a linear regression analysis was performed toassess the relation between the two test systems. The slope andintercept for the regression line were calculated to be 1.0667 and−4.5867, respectively. The regression parameters were used to convertWestern blot titres into infectious titres using the following formula:

Titre_([Bioassay])=(Titre_([WesternBlot])+4.58671/(1.0667).

Once the infectious titre per ml was calculated, the total prion boundto the column was determined. The calculated prion protein bindingcapacity of the PRDT resin is shown in the Table 14 below. The capacitywas determined using the amount of PrPsc observed directly bound to thePRDT resin (samples Reg and Col).

TABLE 14 Prion binding capacity of PRDT Resin Infectious Binding PrPsccapacity Sample ID Sample Description (log₁₀ ID₅₀ total) = per Albuminspiked with a sarkosyl solubilized Total PrP^(sc) Total PrPsc + ml spike(log₁₀ ^(WB))^(#) 4.1 (ID₅₀/ml)* SP0912-SSM 50 ml plasma spiked with 0.5ml 3.7

7.8 — of a sarkosyl solubilized spike 5.0 ml PRDT Resin; 50 ml ofSP0912-SSM sample loaded (Target flow rate: 1.8 ml/min) SP0912-Reg 2MNaCl salt wash (sample ≦1.9 ≦6.0 ≦5.3 log₁₀/ml volume 25.1 ml)SP0912-Col Column resin (sample volume 3.9 8.0   7.3 log₁₀/ml 8.7 ml)^(#)Total PrPSc includes corrections for volume of sample as well asconcentration/dilution prior to testing

Total load based on a final volume of 50 ml loaded onto the PRDT column*Based on a 5.0 ml column size

The total binding of PrPsc to the PRDT matrix following a 2M salt washwas 7.4 log₁₀ ID₅₀/ml. No PrPsc was detected in the salt wash fractionsby Western blot. Significant PrPsc removal (≧2.0 log₁₀) was alsoobserved.

The pH of the spiked starting material was 6.85. After the addition of65 μl 0.1 M NaOH and 50 μl 1 M NaOH the pH value remained unchanged. Theeffect probably resulted from the significant buffering capacity of 25%albumin

When performing interference testing, the starting material was testedundiluted, with centrifugation and 10-fold concentration. This was inaddition to the interference testing described in the study plan, andwas performed to allow an evaluation of the possibility forconcentrating the samples and thus achieving higher reduction factors.

Example 5 In-Process and Stability Analyses for Human Albumin Treatedwith Prion Removal

In this experiment, the effect of prion-removal column on theconcentration and composition of human albumin was evaluated. In twoseparate experiments, one liter of albumin was treated with thePRIOCLEAR™ B column (50 ml) (ProMetic Biosciences). The untreatedsample, sample collected at the beginning of the run, and samplecollected at the end of the run were then subject to concentration andcomposition analyses. The results are shown in Tables 15 and 16.

TABLE 15 Result for Human Albumin in Experiment One HA Concentration, HAComposition (%) Sample Description mg/mL Monomer Dimer Polymer OligomerHA, Untreated 252 93.73 2.71 3.56 ND HA Post Prion-removal Column, 25393.93 2.71 3.36 ND Sample Collected at the Beginning of Run HA PostPrion-removal Column, 255 93.76 2.72 3.52 ND Sample Collected at the Endof Run (1 L)

TABLE 16 Result for Human Albumin in Experiment Two HA Concentration, HAComposition (%) Sample Description mg/mL Monomer Dimer Polymer OligomerHA, Untreated 206 92.15 3.08 4.34 0.44 HA Post Prion-removal Column, 18192.26 3.05 4.27 0.42 Sample Collected at the Beginning of Run HA PostPrion-removal Column, 206 92.19 3.08 4.30 0.43 Sample Collected at theEnd of Run (1 L)

No significant differences on the albumin were observed before and afterthe prion removal process.

We further evaluated the in-process stability of human albumin treatedfor prion-removal and Abraxane® suspensions manufactured using the humanalbumin treated for prion-removal. The result is shown in Tables 17 and18.

TABLE 17 In-process Stability of Treated HA Solutions from ExperimentOne and Abraxane Suspensions Manufactured using Treated HA HA SampleStorage Concentration, HA Composition (%) Description Conditions mg/mLMonomer Dimer Polymer Oligomer Concentrated 0-time 255 93.79 2.73 3.48ND HA Solution 24 hrs at 5° C. 257 93.76 2.74 3.50 ND 48 hrs at 5° C.255 93.74 2.77 3.49 ND 72 hrs at 5° C. 255 93.76 2.73 3.50 ND Diluted HA0-time 51 93.79 2.73 3.47 ND Solution 24 hrs at 5° C. 51 93.82 2.71 3.47ND 48 hrs at 5° C. 51 93.83 2.70 3.47 ND 72 hrs at 5° C. 51 93.85 2.693.46 ND Abraxane ® 0-time 57 88.39 7.14 3.26 1.22 Suspension 24 hrs at25° C. 57 86.32 8.19 3.25 2.25 before 36 hrs at 25° C. 55 88.22 8.442.13 1.21 Lyophilization

TABLE 18 In-process Stability of Treated HA Solutions from ExperimentTwo and Abraxane Suspensions Manufactured using Treated HA HA SampleStorage Concentration, Description Conditions mg/mL HA Composition (%)Sample Storage Concentration, Monomer Dimer Polymer OligomerConcentrated 0-time 206 92.22 3.09 4.25 0.44 HA Solution 24 hrs at 5° C.202.7 92.28 3.03 4.26 0.43 48 hrs at 5° C. 204.1 92.32 2.99 4.25 0.44 72hrs at 5° C. 200.3 92.14 3.06 4.35 0.44 Diluted HA 0-time 51.4 92.283.04 4.26 0.42 Solution 24 hrs at 5° C. 51.1 92.30 3.01 4.27 0.43 48 hrsat 5° C. 52.1 92.38 2.95 4.24 0.43 72 hrs at 5° C. 51.7 92.45 2.93 4.220.41 Abraxane ® 0-time 57 85.78 8.30 3.97 1.94 Suspension 24 hrs at 25°C. 57.1 85.28 8.67 3.95 2.10 before 36 hrs at 25° C. 57.4 87.72 7.623.43 1.23 Lyophilization

We further compared the accelerated stability of human albumin infinished Abraxane® products manufactured using human albumin treated forprion-removal (Pilot plant batch using human albumin treated for Prionremoval) and finished Abraxane® products manufactured using humanalbumin not treated for prion-removal (Abraxane Exhibit lot, AbraxaneValidation lot, and Abraxane pilot plant batch). The result is shown inTable 19.

TABLE 19 Accelerated Stability of HA in Abraxane Finished ProductsManufactured using HA Treated for Prion-removal. Comparison withFinished Products Manufacturing using Untreated HA HA CompositionMonomer Dimer Polymer Oligomer Change Change Change Change SampleStorage During During During During Description Experiments Conditions %Storage % Storage % Storage % Storage Pilot Plant Experiment 1 0-time84.66 N/A 9.33 N/A 2.76 N/A 3.25 N/A Batch using 2 W at 55° C. 70.39−14.27 17.16 7.83 8.77 6.01 3.67 0.42 HA Treated 1 M at 55° C. 61.83−22.83 19.34 10.01  14.55 11.79  4.27 1.02 for Prion 1 M at 40° C. 76.22 −8.44 14.58 5.25 5.71 2.95 3.49 0.24 Removal Abraxane Experiment 10-time 86.70 N/A 9.40 N/A 2.1 N/A 2.50 N/A Exhibit Lot 3 M at 40° C.71.20 −15.50 17.20 7.80 8.9 6.80 2.70 0.20 3 M at 40° C. 63.90 −22.8019.30 9.90 13.9 11.80  3.00 0.50 Pilot Plant Experiment 2 0-time 84.44N/A 9.03 N/A 2.32 N/A 4.17 N/A Batch using 2 W at 55° C. 72.43 −12.0116.02 6.99 7.12 4.80 4.39 0.22 HA Treated for Prion Removal AbraxaneExperiment 2 0-time 86.70 N/A 7.00 N/A 1.5 N/A 4.80 N/A Process 3 M at40° C. 75.60 −11.10 14.30 7.30 5.4 3.90 4.70 −0.10  Validation Lot 6 Mat 40° C. 68.80 −17.90 16.80 9.80 9.2 7.70 5.20 0.40 Abraxane PilotExperiments 0-time 89.45 N/A 5.73 N/A 1.33 N/A 3.5 N/A Plant Batch 1 and2 2 W at 55° C. 75.19 −14.26 14.92 9.19 6.21 4.88 3.68 0.18

Comparison of stability data shows that storage for 2 weeks at 55° C. isequivalent to storage for 3 months at 40° C. No significant differenceswere observed during the manufacturing process and during the in-processtesting between pilot plant batches manufactured using human albumintreated for prion removal and pilot plant batches manufactured usinguntreated human albumin There were no significant differences betweenthe properties of the finished products manufactured using human albumintreated for prion removal and the properties of the finished productmanufactured using untreated human albumin

Example 6 Evaluation of the Effect of Prion-Removal Column on Abraxane®in-Process Suspension

This experiment evaluates the effect of prion removal from Abraxane®in-process suspension, namely, the suspension of Abtaxane® prior tolyophilization. Commercially available PIKSI kit columns (1 cc),containing Toyopearl Amino 650CU resin, ProMetic Biosciences was used inthis experiment.

In two separate experiments, 0.5 L Abraxane® in process suspensioncontaining human albumin were processed through the column The analysisresults are summarized in Table 20.

TABLE 20 Effect of Prion-Removal Column on Abraxane In-ProcessSuspension HA Impurities, % Sample Conc., HA Composition, % Paclitaxel,7- Particle size, nm No. mg/mL Monomer Dimer Polymer Oligomer mg/mLEpipaclitaxel Total Mean <5% <95% <99.9% pH 1 55 88.29 5.95 3.99 1.326.9 0.08 0.25 131 82 193 254 7.0 2 56 88.37 5.96 3.86 1.36 6.9 0.08 0.25132 86 190 250 7.0 3 56 88.38 5.96 3.76 1.39 6.8 0.09 0.26 130 82 190250 7.0

Sample 1: Abraxane In-Process Suspension, Untreated

Sample 2: Abraxane In-Process Suspension, post Prion-removal Column,sample Collected at the Beginning of Run

Sample 3: Abraxane In-Process Suspension, post Prion-removal Column,sample Collected at the End of Run (0.5 L).

As shown in Table 20, the physical and chemical testing of Abraxane®in-process suspension treated for prion removal show no significantdifferences between the untreated and treated suspension, in terms ofparticle size, pH, paclitaxel assay and impurities, human albumin (HA)assay, and HA composition.

1. A composition comprising nanoparticles comprising albumin and asubstantially water insoluble pharmacologically active agent, whereinthe composition is substantially free of a prion protein.
 2. Thecomposition of claim 1, wherein the composition has a prion infectivityof less than about 100 fg/ml.
 3. The composition of claim 1, wherein thecomposition has a prion infectivity of less than about 10 IU-ic/ml. 4.The composition of claim 1, wherein the composition does not show thepresence of a prion protein based on a protein misfolding cyclicamplification (PMCA) assay or based on an IPCR assay.
 5. The compositionof claim 1, further comprising a trace amount of a ligand capable ofbinding to a prion protein.
 6. A composition comprising nanoparticlescomprising albumin and a substantially water insoluble pharmacologicallyactive agent, wherein the albumin in the composition was obtained by amethod comprising a prion removal process, said prion removal processcomprising contacting an initial albumin composition with a ligandcapable of binding to a prion protein.
 7. The composition of claim 6,wherein the prion removal process further comprises removing said ligandand proteins bound thereto from said albumin and composition.
 8. Thecomposition of claim 7, wherein the ligand is a peptide.
 9. Thecomposition of claim 7, wherein the ligand is a trazine-based compound.10. A method of producing a composition comprising nanoparticlescomprising albumin and a substantially water insoluble pharmacologicallyactive agent, said method comprising: a) removing a prion protein froman initial albumin composition; b) subjecting a mixture comprising asolution comprising the prion-removed albumin and an organic phasecomprising said substantially water insoluble pharmacologically activeagent dispersed in an organic solvent to a high shear condition.
 11. Themethod of claim 10, wherein step a) comprises: 1) contacting the initialalbumin solution with a ligand capable of binding to a prion protein.12. The method of claim 11, wherein step a) further comprises: 2)removing the ligand and proteins bound thereto from the albuminsolution.
 13. The method of claim 11, further comprising removing saidorganic solvent from the mixture.
 14. The method of claim 11, whereinsaid ligand is a peptide.
 15. The method of claim 11, wherein the ligandis a triazine-based compound.
 16. The method of claim 10, wherein theinitial albumin composition is a blood derived product.
 17. A method ofproducing a composition comprising nanoparticles comprising albumin anda substantially water insoluble pharmacologically active agent,comprising: a) subjecting a mixture comprising an organic phasecomprising said substantially water insoluble pharmacologically activeagent and an albumin solution to a high shear condition, and b) removinga prion protein from said mixture.
 18. The method of claim 17, whereinstep b) comprises: 1) contacting the mixture with a ligand capable ofbinding to a prion protein.
 19. The method of claim 18, wherein step b)further comprises: 2) removing the ligand and proteins bound theretofrom said mixture.
 20. The method of claim 18, wherein said ligand is apeptide.
 21. The method of claim 18, wherein the ligand is atriazine-based compound.
 22. A method of removing a prion protein from acomposition suspected of containing a prion protein comprisingnanoparticles comprising albumin and a substantially water insolublepharmacologically active agent, comprising: a) contacting thecomposition with a ligand capable of binding to a prion protein, b)removing the ligand and proteins bound thereto from the composition.